The levels of Streptococcus (S.) mutans infections in saliva were evaluated and a comparison for specific antibody levels among children with different levels of S. mutans infection was made. The promising epitopic regions of antigen AgI/II (PAc) and glucosyltransferase (GTF) for potential vaccine targets related to S. mutans adherence were screened. A total of 94 children aged 3-4 years were randomly selected, including 53 caries-negative and 41 caries-positive children. The values of S. mutans and those of salivary total secretory immunoglobulin A (sIgA), anti-PAc and anti-Glucan binding domain (anti-GLU) were compared to determine the correlation among them. It was found the level of s-IgA against specific antigens did not increase with increasing severity of S. mutans infection, and the complete amino acid sequence of PAc and GTFB was analyzed using the DNAStar Protean system for developing specific anti-caries vaccines related to S. mutans adherence. A significantly positive correlation between the amount of S. mutans and children decayed, missing, and filled teeth index was observed. No significant difference was detected in specific sIgA against PAc or GLU between any two groups. No significant correlation was found between such specific sIgA and caries index. A total of 16 peptides from PAc as well as 13 peptides from GTFB were chosen for further investigation. S. mutans colonization contributed to early children caries as an important etiological factor. The level of sIgA against specific antigens did not increase with increasing severity of S. mutans infection in children. The epitopes of PAc and GTF have been screened to develop the peptide-based or protein-based anti-caries vaccines.
Antibodies, Bacterial ; biosynthesis ; Antigens, Bacterial ; chemistry ; immunology ; Bacterial Proteins ; chemistry ; immunology ; Case-Control Studies ; Child, Preschool ; Dental Caries ; immunology ; pathology ; prevention & control ; Epitopes ; chemistry ; immunology ; Female ; Glucosyltransferases ; chemistry ; immunology ; Humans ; Immunoglobulin A, Secretory ; biosynthesis ; Male ; Peptides ; chemistry ; immunology ; Saliva ; chemistry ; microbiology ; Severity of Illness Index ; Streptococcal Vaccines ; biosynthesis ; chemistry ; immunology ; Streptococcus mutans ; chemistry ; immunology ; pathogenicity ; Vaccines, Subunit ; Virulence Factors ; chemistry ; immunology

Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces.
Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; drug effects ; Colony Count, Microbial ; Fatty Acids ; pharmacology ; Humans ; Lipids ; pharmacology ; Microscopy, Electron ; Mouth Mucosa ; chemistry ; immunology ; microbiology ; Porphyromonas gingivalis ; chemistry ; drug effects ; ultrastructure ; Saliva ; chemistry ; microbiology ; Sphingolipids ; pharmacology ; Virulence ; drug effects

Diagnosis of scrub typhus is difficult because its symptoms are very similar to other acute febrile illnesses, such as leptospirosis, murine typhus, and other viral hemorrhagic fevers. To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We have developed a chimeric recombinant antigen cr56 and two other recombinant antigens, r21 and kr56, from various serotypes of Orientia tsutsugamushi. They were tested for the detection of antibodies against O. tsutsugamushi in the patient's serum samples using enzyme-linked immunosorbent assay (ELISA) and dot-blot analyses. As of conventional immunofluorescence assay (IFA), when the mixture of these three recombinant antigens was used, both sensitivity and specificity of the recombinant antigens were increased up to 98% in IgM and IgG at ELISA and dot blotting. Additionally, both sensitivity and specificity by detection of IgM and IgG antibodies at rapid diagnostic test (RDT), using the mixture of three antigens and gold conjugated antibodies, were 99%. Our results suggest the use of mixture of these recombinant antigen proteins in ELISA or RDT is suitable as a diagnostic test for scrub typhus.
Antibodies, Bacterial/blood/chemistry/immunology ; Antigens, Bacterial/diagnostic use/genetics/metabolism ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect ; Gold/chemistry ; Humans ; Immunoassay ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Orientia tsutsugamushi/immunology/*metabolism ; Recombinant Proteins/biosynthesis/diagnostic use/genetics ; Scrub Typhus/*diagnosis ; Sensitivity and Specificity ; Serotyping

As the dominant antigens, early secreted antigenic target 6 (ESAT6, E6) and culture filtrate protein 10 (CFP10, C10) had once been the focus of tuberculosis (TB) vaccine due to their capability of inducing strong cell immune response in the host. They are also endowed with promising future of prevention against and diagnosis of TB. In this review, we systematically introduce recent research progress of E6 and C10, especially in structure-function, biological characteristics, protein expression and secretion, host immunity and vaccine development, and the prospects of their application are also discussed.
Antigens, Bacterial ; chemistry ; genetics ; immunology ; Bacterial Proteins ; chemistry ; genetics ; immunology ; Humans ; Immunodominant Epitopes ; immunology ; Molecular Biology ; Peptide Fragments ; chemistry ; genetics ; immunology ; Tuberculosis Vaccines ; genetics ; immunology ; Vaccines, DNA ; immunology

OBJECTIVETo clone the plasmid protein pORF8 of Chlamydia trachomatis and localize its expression in Chlamydia-infected cells.

METHODSpORF8 gene was amplified and cloned into pGEX-6p vector, and the pORF8 fusion protein was expressed in E.coli XL1 Blue. After purification with glutathione-conjugated agarose beads, the pORF8 fusion protein was used to immunize BALB/c mice to generate polyclonal antibodies against pORF8 protein. The antibodies obtained were used to localize the plasmid protein pORF8 in Chlamydia-infected cells with immunofluorescence assay (IFA).

RESULTSThe pORF8 gene 744 bp in length was successfully cloned and the GST fusion protein with a relative molecular mass of 54 000 was obtained. The cellular distribution pattern of the plasmid protein pORF8 was similar to that of the major outer membrane protein (MOMP), a known C. trachomatis inclusion body protein, but not to that of chlamydial protease-like activity factor (CPAF, a secreted protein).

CONCLUSIONThe plasmid protein pORF8 is localized on the bacterial organism as an inclusion body protein in C. trachomatis-infected cells. The cellular location of pORF8 protein can potentially provide important insights into the pathogenesis of C. trachomatis.

Animals ; Antibodies ; immunology ; Bacterial Proteins ; biosynthesis ; genetics ; Chlamydia Infections ; metabolism ; Chlamydia trachomatis ; chemistry ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; HeLa Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Plasmids ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Corn, one of the most important forage crops worldwide, has proven to be a useful expression vehicle due to the availability of established transformation procedures for this well-studied plant. The exotoxin Apx, a major virulence factor, is recognized as a common antigen of Actinobacillus (A.) pleuropneumoniae, the causative agent of porcine pleuropneumonia. In this study, a cholera toxin B (CTB)-ApxIIA#5 fusion protein and full-size ApxIIA expressed in corn seed, as a subunit vaccine candidate, were observed to induce Apx-specific immune responses in mice. These results suggest that transgenic corn-derived ApxIIA and CTB-ApxIIA#5 proteins are potential vaccine candidates against A. pleuropneumoniae infection.
Actinobacillus Infections/microbiology/*prevention & control ; Actinobacillus pleuropneumoniae ; Animals ; Antigens, Bacterial/immunology ; Bacterial Proteins/*immunology ; Bacterial Vaccines/*immunology ; Cholera Toxin/*chemistry ; Female ; Hemolysin Proteins/*immunology ; Immunization, Secondary ; Mice ; Mice, Inbred ICR ; Plants, Genetically Modified ; Zea mays/*genetics

BACKGROUND: Enzyme immunoassay (EIA) capable of detecting both toxin A and toxin B is strongly recommended for the diagnosis of Clostridium difficile associated disease. Therefore, we evaluated two different EIAs for the detection of C. difficile toxin A/B. METHODS: For a total of 228 stool specimens we performed bacteriologic cultures for C. difficile and examined for toxin A and toxin B using enzyme linked fluorescent immunoassay (ELFA; VIDAS CDAB, Bio-Merieux sa, France) and ELISA (C.DIFFICILE TOX A/B II, TECHLAB, USA). We also performed PCR assays for toxin A and B genes in 117 C. difficile isolates that grew from the stool cultures and compared the results with those obtained with the two different EIAs. RESULTS: The concordance rate between ELFA and ELISA was 85.5% (195/228). Using the culture and PCR results as the standard, the sensitivity/specificity of the ELFA and ELISA were 65.0%/72.1% and 71.8%/70.3%, and for positive/negative predictive values were 78.4%/69.6% and 71.8%/70.3%, respectively (P value >0.05). No differences were observed between the results of ELFA and ELISA with toxin A- toxin B+ strains of C. difficile. CONCLUSIONS: The sensitivity of the ELISA was slightly higher than that of ELFA for toxin A and toxin B, but the specificity and positive predictive value of the ELFA were rather higher than those of the ELISA, although no statistical differences were observed. A bacteriologic culture and PCR assay for toxin genes are recommended in case the both EIAs are negative.
Bacterial Proteins/*analysis/genetics/immunology ; Bacterial Toxins/*analysis/genetics/immunology ; Clostridium difficile/genetics/isolation & purification/*metabolism ; Enterotoxins/*analysis/genetics/immunology ; Enzyme-Linked Immunosorbent Assay/*methods ; Feces/microbiology ; Fluorescent Dyes/chemistry ; Humans ; Reagent Kits, Diagnostic

OBJECTIVETo investigate the immunoregulatory effects of pertussis protein on airway inflammatory, IFN-gamma/IL-4 ratio in bronchoalveolar lavage fluids(BALF) and airway hyperresponsiveness (AHR) in the sensitized mice.

METHODSThe sensitized mice were reexposed to ovalbumin and the airway response to methacholine injection was monitored. Inflammatory cells and cytokines IFN-gamma/IL-4 ratio in BALF were measured. Lung tissue specimens were collected for histological examination.

RESULTIntramuscular injection or intranasal instillation of pertussis protein inhibited changes in lung resistance and lung dynamic compliance, upregulated IFN-gamma/IL-4 ratio and decreased eosinophil accumulation in a dose-dependent manner. Pathological examination showed that goblet cell hyperplasia and inflammatory cells infiltration in lung tissue were suppressed by pertussis protein.

CONCLUSIONPertussis protein inhibits the inflammation and regulates the function of lungs in asthma mice, suggesting its potential application in treatment of asthma.

Albumins ; Animals ; Asthma ; chemically induced ; immunology ; therapy ; Bacterial Proteins ; immunology ; pharmacology ; Bacterial Toxins ; immunology ; pharmacology ; Bronchoalveolar Lavage Fluid ; chemistry ; Interferon-gamma ; analysis ; Interleukin-4 ; analysis ; Male ; Methacholine Chloride ; Mice ; Mice, Inbred ICR

The cp36 gene encoding an adhesive protein was amplified by PCR from genomic DNA of rabbit P. multocida C51-3 strain, and cloned into the pMD18-T vector and then sequenced. The mature adhesive protein without a signal peptide of cpm36 gene was amplified by PCR from the recombinant plasmid pMD18-cp36, then cloned into the prokaryotic expression vector pQE30 to provide a recombinant plasmid pQE30-cpm36. The recombinant protein of CPM36 was produced in Escherichia coli M15 harboring the recombinant plasmid pQE30-cpm36 by IPTG induction, and the recombinant protein purified by the affinity chromatography with Ni(2+)-NTA resin. The sequence analyses showed that the ORF of cp36 gene was 1032 bp in length, and DNA homology of the cp36 genes between the C51-3 strain and the previously reported different serotype strains of P. multocida in GenBank was 76.9 to 100%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 37 kD, and the Western blotting analysis demonstrated that the recombinant protein CPM36 and native 36 kD protein of C51-3 were recognized specifically by an antiserum against the recombinant protein, suggesting that the recombinant protein is an antigenic protein.
Adhesins, Bacterial ; biosynthesis ; genetics ; immunology ; Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Pasteurella multocida ; chemistry ; Rabbits ; microbiology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

An isolated virulence Riemerella anatipestifer strain passaged 200 times on TSB agar were used for the virulent to avirulent conversion. The effects of passage on biological properties of outer membrane proteins (OMPs) were investigated using the virulent and avirulent strains. Transmission electron microscopy demonstrated that the avirulent strain produced lower amounts of outer membrane vesicles and the outer membrane decreased, the cytoplasmic appearance jumbled. The OMPs of the virulent strain agglutinated only in RA serotype 2 antisera, whereas the OMPs of the avirulent strain agglutinated in antisera of RA 1, 2, 10 and 11. SDS-PAGE Analysis showed the OMPs profiles of both strains were similar but the immunoblotting profiles were different. The protective immunity against Riemerella anatipestifer infection was investigated by immunizations with OMPs in ducks. ELISA results showed that the OMPs induced the production of antibodies in immunized ducks, but the OMPs of virulence strain induced higher antibody titers than the attenuated strain (P < 0.05). RA2 group showed significantly higher survival rates (100%) than RA200 group (0%) after challenged with the homologous virulent strain. The ompA gene of both stains were also amplified by PCR, nucleotide homology was 99.9%. In conclusion, OMPs of virulent RA strain are suitable candidates for vaccine development. Biological properties of OMPs undergoes significant changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains.
Animals ; Bacterial Outer Membrane Proteins ; chemistry ; genetics ; immunology ; Ducks ; Flavobacteriaceae ; chemistry ; classification ; immunology ; Flavobacteriaceae Infections ; immunology ; microbiology ; veterinary ; Serotyping