ABSTRACT
Leptospirosis, a zoonotic disease that is caused by many serovars which are more than 200 in the world, is an emerging worldwide disease. Accurate and rapid diagnostic tests for leptospirosis are a critical step to diagnose the disease. There are some commercial kits available for diagnosis of leptospirosis, but the obscurity of a species- or genus-specific antigen of pathogenic Leptospira interrogans causes the reduced sensitivity and specificity. In this study, the polysaccharide derived from lipopolysaccharide (LPS) of nonpathogenic Leptospira biflexa serovar patoc was prepared, and the antigenicity was confirmed by immunoblot and enzyme linked immunosorbent assay (ELISA). The performance of the rapid diagnostic test (RDT) kit using the polysaccharide as a diagnostic antigen was evaluated in Korea, Bulgaria and Argentina. The sensitivity was 93.9%, 100%, and 81.0% and the specificity was 97.9%, 100%, and 95.4% in Korea (which is a rare region occurring with 2 serovars mostly), Bulgaria (epidemic region with 3 serovars chiefly) and Argentina (endemic region with 19 serovars mainly) respectively. These results indicate that this RDT is applicable for global diagnosis of leptospirosis. This rapid and effective diagnosis will be helpful for diagnosis and manage of leptospirosis to use and the polysaccharide of Leptospira may be called as genus specific antigen for diagnosis.
Subject(s)
Female , Humans , Male , Antigens, Bacterial/immunology , Argentina , Bulgaria , Enzyme-Linked Immunosorbent Assay , Leptospira/isolation & purification , Leptospira interrogans/isolation & purification , Leptospirosis/diagnosis , Polysaccharides/immunology , Reagent Kits, Diagnostic/standards , Republic of Korea , Sensitivity and SpecificityABSTRACT
Leptospirosis, a zoonotic disease that is caused by many serovars which are more than 200 in the world, is an emerging worldwide disease. Accurate and rapid diagnostic tests for leptospirosis are a critical step to diagnose the disease. There are some commercial kits available for diagnosis of leptospirosis, but the obscurity of a species- or genus-specific antigen of pathogenic Leptospira interrogans causes the reduced sensitivity and specificity. In this study, the polysaccharide derived from lipopolysaccharide (LPS) of nonpathogenic Leptospira biflexa serovar patoc was prepared, and the antigenicity was confirmed by immunoblot and enzyme linked immunosorbent assay (ELISA). The performance of the rapid diagnostic test (RDT) kit using the polysaccharide as a diagnostic antigen was evaluated in Korea, Bulgaria and Argentina. The sensitivity was 93.9%, 100%, and 81.0% and the specificity was 97.9%, 100%, and 95.4% in Korea (which is a rare region occurring with 2 serovars mostly), Bulgaria (epidemic region with 3 serovars chiefly) and Argentina (endemic region with 19 serovars mainly) respectively. These results indicate that this RDT is applicable for global diagnosis of leptospirosis. This rapid and effective diagnosis will be helpful for diagnosis and manage of leptospirosis to use and the polysaccharide of Leptospira may be called as genus specific antigen for diagnosis.
Subject(s)
Female , Humans , Male , Antigens, Bacterial/immunology , Argentina , Bulgaria , Enzyme-Linked Immunosorbent Assay , Leptospira/isolation & purification , Leptospira interrogans/isolation & purification , Leptospirosis/diagnosis , Polysaccharides/immunology , Reagent Kits, Diagnostic/standards , Republic of Korea , Sensitivity and SpecificityABSTRACT
BACKGROUND: Many strains of Leptospira interrogans have been isolated in Korea since 1984. Most isolates were identified as serovar lai by serological methods. The pulsed field gel electrophoresis (PFGE) patterns of Korean isolates have not been investigated currently. METHODS: 29 reference strains and 29 Korean isolates of Leptospira interrogans were characterized by PFGE. Chromosomes were digested by the Not I restriction enzyme and subsequently PFGE was performed in CHEF-DRII (Bio Rad Lab) with 3 pulse times (30 seconds 13 hours, 60 seconds 13 hours, 120 seconds 14 hours) at 150 V (4.5 V/cm). RESULTS: 12 serogroup reference strains and most 17 serovars reference strains in the serogroup Icterohaemoffhagie showed the unique Not I restriction patterns. Most isolates identified serologically as serovar lai showed the same PFGE patterns as the serovar lai reference strain. The strain HM3 and 18R identified serologically as new serovars yeonchon and hongchon respectively showed the same PFGE patterns as serovar lai. The strain AP31, CH88-19 and NR13 that were different from serovar lai by serological methods showed the PFGE patterns indistinguishable from serovar lai reference strain. The strain HY2 that was identified as serovar lai, and the strain 30R that was different from serovar lai serologically showed the PFGE patterns slightly different from serovar lai reference strain. CONCLUSION: The PFGE profile of most Korean isolates Leptospira interrogans serologically identified as serovar lai is identical to the reference strain serovar lai. PFGE analysis thus may be applied to identify serovar of isolates and to investigate the genetic diversity of related serovar.
Subject(s)
DNA , Electrophoresis, Gel, Pulsed-Field , Genetic Variation , Korea , Leptospira interrogans , LeptospiraABSTRACT
BACKGROUND: Many strains of Leptospira interrogans have been isolated in Korea since 1984. Most isolates were identified as serovar lai by serological methods. The pulsed field gel electrophoresis (PFGE) patterns of Korean isolates have not been investigated currently. METHODS: 29 reference strains and 29 Korean isolates of Leptospira interrogans were characterized by PFGE. Chromosomes were digested by the Not I restriction enzyme and subsequently PFGE was performed in CHEF-DRII (Bio Rad Lab) with 3 pulse times (30 seconds 13 hours, 60 seconds 13 hours, 120 seconds 14 hours) at 150 V (4.5 V/cm). RESULTS: 12 serogroup reference strains and most 17 serovars reference strains in the serogroup Icterohaemoffhagie showed the unique Not I restriction patterns. Most isolates identified serologically as serovar lai showed the same PFGE patterns as the serovar lai reference strain. The strain HM3 and 18R identified serologically as new serovars yeonchon and hongchon respectively showed the same PFGE patterns as serovar lai. The strain AP31, CH88-19 and NR13 that were different from serovar lai by serological methods showed the PFGE patterns indistinguishable from serovar lai reference strain. The strain HY2 that was identified as serovar lai, and the strain 30R that was different from serovar lai serologically showed the PFGE patterns slightly different from serovar lai reference strain. CONCLUSION: The PFGE profile of most Korean isolates Leptospira interrogans serologically identified as serovar lai is identical to the reference strain serovar lai. PFGE analysis thus may be applied to identify serovar of isolates and to investigate the genetic diversity of related serovar.
Subject(s)
DNA , Electrophoresis, Gel, Pulsed-Field , Genetic Variation , Korea , Leptospira interrogans , LeptospiraABSTRACT
Leptospirosis has been one of important epidemic diseases in Korea since 1984. Wild rodents, mostly Apodemus agrarius, served the important source of infection especially in harvest season in rural areas of Korea. Prevalence of Leptospira spp. infection in field rodents were investigated by detecting leptospiral DNA from rodent kidney. Among 108 rodents collected from various areas in 1998, leptospiral DNA were detected from 7 rodents (6.48%). Among 104 rodents, Apodemus agrarius, captured from Yeonchon and Paju area in 2001 and 2002, leptospiral DNA were detected from 6 rodents (5.76%). No leptospiral DNA was detected from 23 rodonts (Apodemus peninsulae 16, Apodemus agrarius 2 and Eothenomys regulus 5) captured in Odae mountain area in 1998.
Subject(s)
Animals , DNA , Kidney , Korea , Leptospira , Leptospirosis , Murinae , Polymerase Chain Reaction , Prevalence , Rodentia , SeasonsABSTRACT
Murine typhus is an acute febrile illness caused by Rickettsia typhi. It is one of the four major acute febrile illnesses in Korea during autumn. To study a species-specific antigen of R. typhi, two clinical isolates (87-91 and 87-100) and two reference strains (VR-144 and VR-738) were analyzed by mouse antisera and monoclonal antibodies (MAbs). On SDS- polyacrylamide gel electrophoresis (PAGE), R. typhi showed major antigen bands of 135, 80, 75, 64, 47, 22, and 19 kDa and these bands differed with those of other species. On Western blot analysis, the MAbs reacting only with R. typhi could only detect 135 kDa protein. The 135 kDa protein appeared to be the species-specific antigen. Other MAbs showing cross-reactivity with R. prowazekii reacted with 135 kDa protein in fresh culture supernatant of R. typhi infected host cell. However, the cross-reacting antibody did also react with smaller protein bands, most of which seem to be degradation products of the 135 kDa protein since they increase in old protein stocks purified from R. typhi harvested from infected host cell. These suggest that 135 kDa protein is unstable and the R. typhi specific epitopes are located at the regions of 135 kDa protein that are removed when the protein is degraded. The 135 kDa protein or its specific and stable recombinant protein would serve an important target for the development of vaccine and specific diagnostic antigen.
Subject(s)
Animals , Mice , Antibodies, Monoclonal , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Epitopes , Immune Sera , Korea , Rickettsia typhi , Rickettsia , Typhus, Endemic Flea-BorneABSTRACT
Serovars of 22 leptospiral field isolates from rats trapped in Korea were identified by cross-agglutinin absorption test (CAAT). Genomic characteristics of 7 selected isolates and 6 antigenically closely related reference serovars of lai, yeonchon, birkini, gem, mwogolo, and canicola were differentiated by arbitrarily primed PCR (AP-PCR) and southern blot hybridization using 16S rRNA gene probe from Borrelia burgdorferi. Among the 22 isolates, 21 strains were identified as serovar lai by CAAT, while the serological reactivity of NR13 did not accord with that of serovar lai. Results of AP-PCR using primers RSP, KF and PB-1 were in general agreement with those obtained by serological identification, and all 7 isolates including NR13 showed the same profile with serovar lai or yeonchon. In the southern blot hybridization with 16S rRNA gene probe, the isolates were divided into two ribotype groups when HindIII and BamHI digests were employed: isolates NR4, NR13, and serovar lai showed the same profile, and isolates JR34, JR57, KR48, JR77, and JR82 were classified as the another ribotype group. Isolate NR13 and serovar yeonchon, which were isolated in Korea and showed serological differences with serovar lai, were indistinguishable from serovar lai in this DNA study using AP-PCR and ribotyping. These results demonstrate that Korean leptospiral isolates were closely related in DNA level, and ribotyping would be useful for subgrouping of field isolates.
Subject(s)
Animals , Rats , Absorption , Blotting, Southern , Borrelia burgdorferi , DNA , Genes, rRNA , Korea , Leptospira , Polymerase Chain Reaction , RibotypingABSTRACT
BACKGROUND: Several studies on the efficacy and safety of the hepatitis B vaccine have shown variable immunogenicity. In this study we reexamined the immunogenicity and safety of recombinant hepatitis B vaccine, Engerix B which have currently been administered to the children in Korea. METHODS: Serum samples were collected from 126 children and 111 adults who were immunized according to the 0, 1, 2-month and 0, 1, 6-month vaccination schedule. Anti-HBs antibody titers were measured by ELISA in sera obtained after each immunization, and compared by immunization schedules. RESULTS: In 62 children with 0, 1, 2-month immunization schedule seroconversion rate was 83.9% after 1st vaccination, 96.8% after 2nd, and 98.4% after 3rd. In 64 children with 0, 1, 6-month immunization schedule seroconversion rates was 78.1% after 1st vaccination, 87.5% after 2nd and 100% after 3rd. In 50 adults immunized with 0, 1, 2-month schedule seroconversionrates was 48.0% after 1st vaccination, 74.0% after 2nd and 90.0% after 3rd. In 61 adults immunized with 0, 1, 6-month schedule seroconversion rate was 44.3% after 1st vaccination, 65.6% after 2nd and 93.4% after 3rd. Seroconversion rate after 0, 1, 2- month vaccination schedule were 98.4% in children and 90.0% in adults. Seroconversion rate after 0, 1, 6-month schedule were 100% in children and 93.4% in adults. There were no significant local and systemic untoward reactions among vaccinees. CONCLUSION: The recombinant Engerix B is excellent in immunogenicity with 93.4% and 100% seroconversion rates in adults and children, respectively. There is no significant difference in seroconversion rate between two vaccination schedule. The vaccine is safe.
Subject(s)
Adult , Child , Humans , Appointments and Schedules , Enzyme-Linked Immunosorbent Assay , Hepatitis B Vaccines , Hepatitis B , Hepatitis , Immunization , Immunization Schedule , Korea , VaccinationABSTRACT
Human papillomavirus (HPV) 16, E7 proteins derived from the prototype (Bac73) and natural variant (Bac101) E7 open reading frame were produced in Sf9 insect cells. The variant E7 gene occurred naturally by substitution mutation at the position of 88 nucleotide, resulting serine instead of asparagine. Using E7 specific monoclonal antibody (VD6), both E7 proteins were identified in recombinant baculovirus infected SF9 cells. Radiolabelling and immunoprecipitation analysis revealed that both E7 proteins were phosphoproteins. Immunostaining result showed that E7 proteins were mainly localized in the cytoplasm. Nuclear form of E7 proteins was also detected after a sequential fractionation procedure for removing chromatin structure. Considering that the VD6 recognition site in E7 protein is located within 10 amino acid at the N-terminus, this region appears to be blocked by the nuclear component. Western blot analysis revealed that nuclear form was more abundant than cytoplasmic E7 proteins. Time course immunostaining showed that the primary location of E7 protein was the nucleus and exported to the cytoplasm as proteins were accumulated. These events occurred similarly in both Bac73 and Bac101 infected Sf9 cells, suggesting that these two proteins may have similar biological functions.
Subject(s)
Humans , Asparagine , Baculoviridae , Blotting, Western , Chromatin , Cytoplasm , Immunoprecipitation , Insecta , Open Reading Frames , Phosphoproteins , Serine , Sf9 CellsABSTRACT
The gene encoding E7 oncoprotein of human papillomavirus type 16 was cloned and expressed in Escherichia coli, and the monoclonal antibodies (Mabs) against this expressed protein were generated. For the efficient immunization, two kind of recombinant E7 protein in fusion form were produced. One was maltose binding protein (MBP) fusion type (MBP-E7) and the other was T7 phage gene 10 product fusioa type (gene 10-E7). Immunization with these two fusion protein to mice, finally two Mabs (VD6 and IB10) were obtained. VD6 and IB10 showed reactivities with E7 protein in CaSki cell but not in HeLa by Western blot analysis. In addition, the Mab, VD6, reacted with COS-7 cell transfected with E7 gene majorly in cytoplasm by immunofluorescence test. Also VD6 could detect E7 protein in cytoplasm and nucleus of CaSki ceU by immunogold electron microscopy. Based on these results, the Mab VD6 was could be used for various E7 detection system such as Western blot analysis and immunohistochemical methods.
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , Bacteriophage T7 , Blotting, Western , Clone Cells , COS Cells , Cytoplasm , Escherichia coli , Fluorescent Antibody Technique , Immunization , Maltose-Binding Proteins , Microscopy, ElectronABSTRACT
No abstract available.
Subject(s)
Electrophoresis, Polyacrylamide Gel , Immunoblotting , Korea , Rickettsia typhi , RickettsiaABSTRACT
No abstract available.
Subject(s)
Humans , Asian People , Hepatitis B Surface Antigens , Hepatitis B , Hepatitis , KoreaABSTRACT
No abstract available.