BACKGROUND: Salmonella is an important pathogen that causes gastroenteritis and sepsis in humans. Recently, changes in serotype prevalence and an increase in antimicrobial resistance have been reported. This study investigated the distribution of Salmonella serotypes and determined the antimicrobial susceptibility of various strains. METHODS: We collected 113 Salmonella isolates other than Salmonella serotype Typhi from 18 university hospitals in 2015. The serotypes were identified by Salmonella antisera O and H according to the Kauffman White scheme. Antimicrobial susceptibility tests for 12 antibiotics were performed using the disk diffusion method or E-test. RESULTS: We identified 22 serotypes. Serotype group B (44.2%) was the most common, followed by groups C (34.5%) and D (21.2%). Salmonella I 4,[5],12:i:- (23.0%), S. Enteritidis (16.8%), and S. Typhimurium (12.4%) were the most common species. Resistance rates for ampicillin, chloramphenicol, ceftriaxone, and trimethoprim/sulfamethoxazole were 46.9%, 18.5%, 8.8%, and 5.3%, respectively. The intermediate resistance rate to ciprofloxacin was 29.2%. Six isolates were extended-spectrum β-lactamase (ESBL) producers, including 5 bla(CTX-M-15) and 1 bla(CTX-M-55). CONCLUSION: There have been changes in the serotype prevalence and antimicrobial resistance of Salmonella in Korea, with a high prevalence of CTX-M 15-positive strains. Continuous monitoring of Salmonella serotypes and antimicrobial resistance is warranted.
Ampicillin ; Anti-Bacterial Agents ; Ceftriaxone ; Chloramphenicol ; Ciprofloxacin ; Diffusion ; Gastroenteritis ; Hospitals, University ; Humans ; Immune Sera ; Korea ; Methods ; Prevalence ; Salmonella ; Sepsis ; Serogroup ; Serotyping

BACKGROUND: Various pneumococcal vaccines have been evaluated for immunogenicity by opsonophagocytic assay (OPA). A multiplexed OPA (MOPA) for 13 pneumococcal serotypes was developed by Nahm and Burton, and expanded to 26 serotypes in 2012. The development of new conjugate vaccines with increased valence has necessitated expanded MOPAs to include these additional serotypes. In this study, we validated this expanded MOPA platform and applied to measure antibodies against 11 additional serotypes (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20B, 22F, and 33F) in human sera. METHODS: All materials, including serum, complement, bacterial master stocks, and HL-60 cells, were evaluated for assay optimization. Following optimization, the assay was validated for accuracy, specificity, and intra- and inter-assay precision with sera from adult donors following standard protocols. The assay was applied to evaluate functional antibodies of 42 sera immunized with 23-valent pneumococcal polysaccharide vaccine (PPV23). RESULTS: The expanded MOPA platform was specific for all serotypes, with the exception of serotype 20. The assay results were highly correlated with those obtained from single-serotype OPA, indicating acceptable accuracy. The coefficients of variation were 7%–24% and 13%–39% in tests of intra- and inter-assay precision, respectively, using three quality-control samples. A MOPA that included 11 additional serotypes in the PPV23 was established and validated with respect to accuracy, specificity, and precision. The opsonic indices of immune sera were obtained using this validated assay. CONCLUSION: The expanded MOPA will be useful for evaluation of the immunogenicity of PPV23 and future conjugate vaccine formulations.
Adult ; Antibodies ; Biological Assay ; Complement System Proteins ; HL-60 Cells ; Humans ; Immune Sera ; Opsonin Proteins ; Pneumococcal Vaccines* ; Sensitivity and Specificity ; Serogroup* ; Tissue Donors ; Vaccines, Conjugate

PURPOSE: There is a need to broaden protective coverage of M protein–based vaccines against group A streptococci (GAS) because coverage of the current 30-valent M protein vaccine does not extend to all emm types. An additional GAS antigen and virulence factor that could potentially extend vaccine coverage is M-related protein (Mrp). Previous work indicated that there are three structurally related families of Mrp (MrpI, MrpII, and MrpIII) and peptides of all three elicited bactericidal antibodies against multiple emm types. The purpose of this study was to determine if a recombinant form containing Mrp from the three families would evoke bactericidal antiserum and to determine if this antiserum could enhance the effectiveness of antisera to the 30-valent M protein vaccine. MATERIALS AND METHODS: A trivalent recombinant Mrp (trMrp) protein containing N-terminal fragments from the three families (trMrp) was constructed, purified and used to immunize rabbits. Anti-trMrp sera contained high titers of antibodies against the trMrp immunogen and recombinant forms representing MrpI, MrpII, and MrpIII. RESULTS: The antisera opsonized emm types of GAS representing each Mrp family and also opsonized emm types not covered by the 30-valent M protein–based vaccine. Importantly, a combination of trMrp and 30-valent M protein antiserum resulted in higher levels of opsonization of GAS than either antiserum alone. CONCLUSION: These findings suggest that trMrp may be an effective addition to future constructs of GAS vaccines.
Antibodies ; Humans ; Immune Sera ; Peptides ; Rabbits ; Staphylococcal Protein A* ; Streptococcal Vaccines* ; Streptococcus pyogenes ; Vaccines ; Virulence ; Virulence Factors

Severe fever with thrombocytopenia syndrome (SFTS) caused by the SFTS virus (SFTSV), a phlebovirus in the family Bunyaviridae, is an emerging tick-borne infectious disease that impacts humans. This disease manifests as a decreased blood cell count and multi-organ failure, with a case-fatality rate of more than 12% in China. Because vaccines or antiviral drugs for the treatment of this disease are not available, monitoring the SFTS circulation in animals and controlling the tick-mammal cycle are important for preventing SFTS. Monoclonal antibodies against the recombinant nucleoprotein of SFTSV were generated to develop a competitive enzyme-linked immunosorbent assay (cELISA) for the detection of antibodies against SFTSV infection in cattle. The specificity and sensitivity of cELISA was assessed by comparing the results of this assay to those of an immunofluorescence assay (IFA). The results of the cELISA using 416 field bovine serum samples and laboratory-immunized positive sera showed 98.1% consistency with those of the IFA. The cELISA used in this study did not show cross-reactivity with antisera against other viral cattle diseases. The cELISA presented in this study can be applied to detect antibodies against SFTSV in cattle.
Animals ; Antibodies ; Antibodies, Monoclonal ; Antiviral Agents ; Blood Cell Count ; Bunyaviridae ; Cattle ; Cattle Diseases ; China ; Communicable Diseases ; Diagnosis* ; Enzyme-Linked Immunosorbent Assay* ; Fever* ; Fluorescent Antibody Technique ; Humans ; Immune Sera ; Nucleoproteins ; Phlebovirus ; Sensitivity and Specificity ; Thrombocytopenia* ; Vaccines

PURPOSE: In the celery-mugwort-birch-spice syndrome, a significant proportion of IgE is directed against high molecular weight (HMW) glycoproteins, including the celery allergen Api g 5. BIP3, a monoclonal antibody originally raised against birch pollen, recognizes HMW allergens in birch and mugwort pollens, celery, and Apiaceae spices. Our aim was to generate mimotopes using BIP3 for immunization against the HMW allergens relevant in the celery-mugwort-birch-spice cross reactivity syndrome. METHODS: Mimotopes were selected from a random-peptide display library by BIP3 and applied in IgE inhibition assays. The 3 phage clones with the highest inhibitory capacity were chosen for immunization of BALB/c mice. Mouse immune sera were tested for IgG binding to blotted birch pollen extract and used for inhibiting patients' IgE binding. Furthermore, sera were tested for binding to Api g 5, to horseradish peroxidase (HRP) as a second glycoprotein, or to non-glycosylated control allergen Phl p 5 in ELISA, and the specific Api g 5-specific IgG titers were determined. RESULTS: Three rounds of biopanning resulted in phage clones exhibiting 7 different sequences including 1 dominant, 1-6-cyclo-CHKLRCDKAIA. Three phage clones had the capacity to inhibit human IgE binding and induced IgG to the HMW antigen when used for immunizing BALB/c mice. The induced BIP3-mimotope IgG reached titers of 1:500 specifically to Api g 5, but hardly reacted to glycoprotein HRP, revealing a minor role of carbohydrates in their epitope. CONCLUSIONS: The mimotopes characterized in this study mimic the epitope of BIP3 relevant for Api g 5, one of the cross-reactive HMW allergens relevant in the celery-mugwort-birch-spice syndrome. BIP3 mimotopes may be used in the future for hyposensitization in this clinical syndrome by virtue of good and specific immunogenicity.
Allergens ; Animals ; Apiaceae ; Apium graveolens ; Artemisia ; Bacteriophages ; Betula ; Carbohydrates ; Clone Cells ; Enzyme-Linked Immunosorbent Assay ; Food Hypersensitivity ; Glycoproteins ; Horseradish Peroxidase ; Humans ; Immune Sera ; Immunization ; Immunoglobulin E ; Immunoglobulin G ; Mice ; Molecular Weight ; Pollen ; Spices ; Vaccination ; Virtues

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na+ and H+ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca2+. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.
Animals ; Cell Line ; Immune Sera/genetics/immunology/*metabolism ; Male ; Mice ; Protozoan Proteins/genetics/*metabolism ; Rabbits ; Recombinant Proteins/immunology ; Sheep ; Sodium-Hydrogen Antiporter/genetics/immunology/*metabolism ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/parasitology/prevention & control

PURPOSE: The Japanese encephalitis virus (JEV) genotype circulating in Korea has changed from G3 to G1. Therefore, the purpose of this study was to compare the antigenic relationship between the two genotypes by using antibody tests. MATERIALS AND METHODS: Blood samples from 42 sows and 216 horses were collected, and their seroprevalence was monitored using the hemagglutination inhibition and virus neutralization tests. Antisera against JEV G1 and G3 were isolated and prepared from guinea pigs. The cross-reactivity of these two viruses was then compared using the neutralizing antibody test. RESULTS: We found that there was a difference in the seropositive ratios of JEV G1 and G3. However, the difference was dependent on the antibody test used. There was also an observed difference in the antigenicity between the two genotypes, as ascertained using the neutralizing antibody test. CONCLUSION: There is an evident difference in JEV antigenicity between the genotypes G1 and G3. Therefore, we propose monitoring of the seroprevalence of JEV, and reevaluating the antigenicity of the current vaccine by using the relevant tests.
Animals ; Antibodies, Neutralizing ; Asian Continental Ancestry Group* ; Cross Reactions ; Encephalitis Virus, Japanese* ; Encephalitis, Japanese* ; Genotype ; Guinea Pigs ; Hemagglutination ; Horses ; Humans ; Immune Sera ; Korea ; Neutralization Tests ; Seroepidemiologic Studies

To compare immunological characteristics of Extracellular fibrinogen-binding protein (Efb) and Clumping factor A (CfA) of Staphylococcus aureus, we constructed two prokaryotic expression vector pET28a-Efb and pET28a-ClfA. After prokaryotical expression and purification, Efb and ClfA were used to immunize experimental animal. After the second immunization the antisera were collected and the antibody titers, the bacteria binding activity and adhesion inhibition activity of these antisera were detected by enzyme linked immunosorbent assay, adhesion inhibition assay and challenge. Both Efb and ClfA had Fibrinogen binding activity whereas the former had better Fibronectin binding activity. The bacteria binding capability of antisera of rabbits immunized with ClfA was better than that with Efb (P < 0.01). Both antisera of Efb and ClfA could inhibit adherence activity of Staphylococcus aureus to Fibrinogen and Fibronectin adherence compare to the control group (P < 0.01), and Efb had better adhesion inhibition activity than ClfA. The antibody titer of immunized group could reach 1:40 500. After the second immunization, both Efb and ClfA had good protective efficacy. This result constitutes a good foundation for Staphylococcus aureus subunit vaccine development.
Animals ; Antibodies, Bacterial ; blood ; Bacterial Adhesion ; Bacterial Proteins ; immunology ; Cattle ; microbiology ; Coagulase ; immunology ; Enzyme-Linked Immunosorbent Assay ; Fibrinogen ; metabolism ; Genetic Vectors ; Immune Sera ; immunology ; Immunization ; Rabbits ; Staphylococcal Infections ; immunology ; Staphylococcus aureus

To generate an epitope-mutated foot-and-mouth disease virus (FMDV) as a marker vaccine, the infectious clone pAsia 1-FMDV containing the complete genomic cDNA of Asia 1 type FMDV was used as backbone, the residues at positions 27 and 31 in the 3D gene were mutated (H27Y and N31R). The resulting plasmid pAsia 1-FMDV-3DM encoding a mutated epitope was transfected into BHK-21 cells and the recombinant virus rAsia 1-3DM was rescued. The recombinant virus showed similar biological characteristics comparable with the parental virus. In serological neutralization test the antisera against recombine virus have a good reactivity with parental virus. The antisera against the mutant virus were shown to be reactive with the mutated epitope but not the wild-type one. The results indicated that the two virus strains could be distinguished by western blotting using synthetic peptides. This epitope-mutated FMDV strain will be evaluated as a potential marker vaccine against FMDV infections.
Animals ; Cell Line ; DNA, Complementary ; Epitopes ; genetics ; Foot-and-Mouth Disease Virus ; genetics ; Immune Sera ; Neutralization Tests ; Transfection

New epidemic broke out in recent year which was suspected to be caused by variant Muscovy duck parvovirus (MDPV). For this reason, new MDPV detection methods are needed for the new virus strains. In this study, a pair of primers were designed according to the full-length genome of MDPV strain SAAS-SHNH, which were identified in 2012, and were used to amplify the vp3 gene of MDPV by polymerase chain reaction. After being sequenced, the vp3 gene was subcloned into the prokaryotic expression vector PET28a. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. SDS-PAGE and Western blotting analysis showed the MDPV vp3 gene was successfully expressed. After being purified by Ni2+ affinity chromatography system, the recombinant protein was used as antigen to immunize rabbits to obtain antiserum. Western blotting analysis showed that the acquired antiserum could react specifically with VP3 protein of J3D6 strain and MDPV vaccine strain. The antiserum could also be used for detection of cultured MDPV from primary duck embryo fibroblasts by immune fluorescence assay (IFA). It could be concluded that the VP3 protein and its antibody prepared in the research could be used for detection of VP3 antiserum and antigen respectively.
Animals ; Blotting, Western ; DNA Primers ; Ducks ; virology ; Electrophoresis, Polyacrylamide Gel ; Immune Sera ; biosynthesis ; Parvovirus ; Polymerase Chain Reaction ; Rabbits ; Recombinant Proteins ; genetics ; Viral Proteins ; genetics