Intranasal Vaccination with OuterMembrane Protein of Orientia tsutsugamushi induces Protective Immunity Against Scrub Typhus

Scrub typhus develops after the individual is bitten by a trombiculid mite infected with Orientia tsutsugamushi. Since it has been reported that pneumonia is frequently observed in patients with scrub typhus, we investigated whether intranasal (i.n.) vaccination with the outer membrane protein of O. tsutsugamushi (OMPOT) would induce a protective immunity against O. tsutsugamushi infection. It was particular interest that when mice were infected with O. tsutsugamushi, the bacteria disseminated into the lungs, causing pneumonia. The i.n. vaccination with OMPOT induced IgG responses in serum and bronchoalveolar lavage (BAL) fluid. The anti-O. tsutsugamushi IgA Abs in BAL fluid after the vaccination showed a high correlation of the protection against O. tsutsugamushi. The vaccination induced strong Ag-specific Th1 and Th17 responses in the both spleen and lungs. In conclusion, the current study demonstrated that i.n. vaccination with OMPOT elicited protective immunity against scrub typhus in mouse with O. tsutsugamushi infection causing subsequent pneumonia.

Serologic Survey and Risk Factors for Coxiella burnetii Infection among Dairy Cattle Farmers in Korea

BACKGROUND: The zoonotic disease Q fever is caused by Coxiella burnetii and usually affects high-risk human populations. We conducted a serological survey of dairy cattle farmers in Korea to determine seroreactivity and identify risk factors for C. burnetii infection. METHODS: This cross-sectional study included 1,824 of 7,219 dairy cattle farms (25.3%) in the study region. The selected dairy cattle farmers visited the nearest public health centers or branches with completed questionnaires. Serum samples from the farmers were tested using an indirect immunofluorescence assay to detect phase II C. burnetii immunoglobulin (Ig) G or M antibodies. RESULTS: A total of 1,222 dairy cattle farmers from 784 dairy cattle farms (43.0%) participated in this study, and 11.0% (134/1,222) exhibited seroreactivity, defined as a phase II antigen IgG or IgM titer ≥ 1:16. In the multivariate analysis, male sex, residence in Gyeonggi Province, a larger herd size, and ocular/oral contact with birth products during calf delivery were significantly associated with a higher risk of C. burnetii infection. Furthermore, the risk was significantly lower among farmers who always wore protective gloves while cleaning cattle excretion, compared to those who sometimes or rarely wore protective gloves. CONCLUSION: Dairy cattle farmers should exercise caution by avoiding ocular/oral contact with birth products during calf delivery and by using protective equipment (including gloves).

Seroreactivity to Q Fever Among Slaughterhouse Workers in South Korea / 예방의학회지

OBJECTIVES: Q fever is a zoonotic disease that occurs worldwide; however, little is known about its prevalence in South Korea. We attempted to determine the prevalence of Q fever seroreactivity among Korean slaughterhouse workers and the risk factors for seroreactivity according to the type of work. METHODS: The study was conducted among 1503 workers at a total of 73 slaughterhouses and 62 residual-product disposal plants. During the study period, sites were visited and surveys were administered to employees involved in slaughterhouse work, and serological tests were performed on blood samples by indirect immunofluorescence assays. Serological samples were grouped by job classification into those of slaughter workers, residual-product handlers, inspectors and inspection assistants, and grading testers and testing assistants. Employee risk factors were analyzed according to the type of work. RESULTS: Out of 1481 study subjects who provided a blood sample, 151 (10.2%) showed reactive antibodies. When these results were analyzed in accordance with the type of work, the result of slaughter workers (11.3%) was similar to the result of residual-product handlers (11.4%), and the result of inspectors and assistants (5.3%) was similar to the result of grading testers and assistants (5.4%). Among those who answered in the affirmative to the survey question, “Has there been frequent contact between cattle blood and your mouth while working?” the proportions were 13.4 and 4.6%, respectively, and this was identified as a risk factor that significantly varied between job categories among slaughterhouse workers. CONCLUSIONS: This study found a Q fever seroreactivity rate of 10.2% for slaughterhouse workers, who are known to be a high-risk population. Contact with cattle blood around the mouth while working was the differential risk factor between job categories among slaughterhouse workers.

Taxonomic Identification of Bacillus Species Using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry / 대한임상미생물학회지

BACKGROUND: In this study, we compared various methods of taxonomic identification of Bacillus strains: biochemical methods, 16S rRNA gene sequencing, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). We also developed a pathogen- isolate resource database, thus increasing the identification rate when using MALDI-TOF MS. METHODS: Thirty Bacillus strains were obtained from the NCCP (National Culture Collection for Pathogens) and were identified using the VITEK 2 system (bio-Mérieux, France), API kit (bioMérieux, France), 16S rRNA gene sequencing, and MALDI-TOF MS. The pathogenicity of Bacillus cereus was confirmed through the identification of virulent genes using a multiplex PCR, and both protein extraction for protein profiling in MALDI-TOF MS and repetitive-sequence fingerprinting were performed. RESULTS: The identification rates at the species level were 40%, 80%, and 76.3% for the VITEK 2 system (bioMérieux), 16S rRNA gene sequencing, and MALDI-TOF MS, respectively. When the major spectrum-profiling dendrogram was compared with the phylogenetic tree, which was constructed based on the 16S rRNA gene sequences and rep-PCR fingerprinting, the classifications were confirmed to be effective. CONCLUSION: Identification of Bacillus strains using MALDI-TOF MS was more effective than that using the VITEK 2 system (bioMérieux), but was similar to that using 16S rRNA gene sequencing. Continual addition to a proteome-based database can result in increased identification rates for MALDI-TOF MS.

Resource Development and Investigation of Novel Species from Unidentified Pathogens in NCCP using MALDI-TOF MS and 16S rRNA Gene Analysis

Species identification is an important item to characterize unidentified bacterial pathogens in developing and managing bacterial resources. In this study, unidentified pathogens based on the results of an automated identification system were identified using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALD-TOF MS) and 16S rRNA gene analysis for development of national resources in the National Culture Collection for Pathogens (NCCP) in Korea. A total of 437 unidentified strains from branch banks of the NCCP were collected, and 16S rRNA and dnaJ gene sequencing, as well as MALDI-TOF MS analysis were performed to identify bacterial species. The mass spectra extracted were analyzed. Twelve strains exhibiting less than 98.65% similarity in 16S rRNA gene were selected as the primary candidates for novel species, and 21 strains exhibiting 98.65~99.0% similarity in 16S rRNA gene were selected as possible candidates for novel species. Among them, strain 32, belonging to Dermabacter sp., was finally selected as a possible strain representing a novel species and 14 unidentified bacterial strains using automated phenotypic identification system were newly registered at NCCP. The present study showed that unidentified pathogens using the automated phenotypic identification system were efficiently identified using the combination of MALDI-TOF MS and 16S rRNA gene analysis, and developed to the national resources in NCCP.

Isolation and Genetic Characterization of Orientia tsutsugamushi from Scrub Typhus Patients in Gyeongsangnam-do, Korea

Orientia tsutsugamushi (O. tsutsugamushi), which is endemic to an Asia-Pacific region, has increased its incidence and caused annually around 10 thousand patients infected with scrub typhus in Korea in the past several years. In the present study, we isolated 44 O. tsutsugamushi from the patients with febrile illness accompanied with or without an eschar in Gyeongsangnam-do, Korea. These isolates were characterized by genetic analysis of the major outer membrane protein, the 56-kDa type-specific antigen (tsa56), which is unique to O. tsutsugamushi. Two types of sequences of tsa56, designated by JJ1 and JJ2, were determined from 37 and 7 isolates of the 44 isolates, respectively. JJ1 and JJ2 showed 74.7~90.8% identity in nucleotide sequence and 66.1~90.5% identity in amino acid sequence with 33 reference strains except for Boryong and Kuroki. JJ1 and JJ2 had 100 and 99.9% nucleotide identity to Boryong strain, and 99.9 and 99.8% to Kuroki, which has been known to be similar to Boryong, respectively. In addition, they showed 77.9~ 81.4% nucleotide identity with the cluster of Gilliam-related genotypes, whereas they showed higher nucleotide identity (89.6~90.8%) with the cluster of Karp-related genotypes. To our knowledge, this is the first report to isolate O. tsutsugamushi and characterize their genotype as the Boryong in Jinju and West Gyeongsangnam-do, Korea, even though it has been reported that the Boryong was the predominant genotype in isolates from chiggers, domestic rodents, and patients in the southern part of Korea. Furthermore, our isolates could be useful source to study on the pathophysiology and epidemiology of scrub typhus in Korea.

Chemoprophylaxis and Serological Follow-Up in Brucella abortus Exposed Laboratory Workers

The aerozolization is one of possible Brucella transmission mechanisms, particularly in air-borne exposed laboratory workers. In this study, seven laboratory workers were potentially exposed to B. abortus via aerosols. Two laboratory workers who sniffed an agar plate several times were considered to be at high risk for acquiring the disease, 5 workers who did not work directly with the strain were be considered at low risk of infection. Prophylactic antibiotics of doxycycline 200 mg/day and rifampin 600 mg/day were offered for 6 weeks in high risk workers and 3 weeks for low risk workers, respectively. Enrolled workers were advised to check for serological testing of Brucella species every 3 weeks during a total period of 12 weeks. Compliance with taking medicine was 57.1% (4/7) and compliance for completing the serological tests was 85.7% (6/7). None of the laboratory workers developed clinical disease or tested positive serologically during 3 months of serological testing and 1 year of clinical follow-up.

Chemoprophylaxis and Serological Follow-Up in Brucella abortus Exposed Laboratory Workers

The aerozolization is one of possible Brucella transmission mechanisms, particularly in air-borne exposed laboratory workers. In this study, seven laboratory workers were potentially exposed to B. abortus via aerosols. Two laboratory workers who sniffed an agar plate several times were considered to be at high risk for acquiring the disease, 5 workers who did not work directly with the strain were be considered at low risk of infection. Prophylactic antibiotics of doxycycline 200 mg/day and rifampin 600 mg/day were offered for 6 weeks in high risk workers and 3 weeks for low risk workers, respectively. Enrolled workers were advised to check for serological testing of Brucella species every 3 weeks during a total period of 12 weeks. Compliance with taking medicine was 57.1% (4/7) and compliance for completing the serological tests was 85.7% (6/7). None of the laboratory workers developed clinical disease or tested positive serologically during 3 months of serological testing and 1 year of clinical follow-up.

Cytokine response in Balb/c mice infected with Francisella tularensis LVS and the Pohang isolate

We investigated the immune response induced by the Francisella (F.) tularensis live vaccine strain (LVS) and the Pohang isolate. After the Balb/c mice were infected intradermally (i.d) with 2 x 10(4) cfu of F. tularensis LVS and Pohang, respectively, their blood and organs were collected at different times; 0, 3, 6, 24, 72, 96, 120 and 168 h after infection. Using these samples, RT-PCR and ELISA analysis were carried out for the comparative study of the cytokines, including TNF-alpha, INF-gamma, IL-2, IL-4, IL-10 and IL-12. In the Pohang-infected mice at 120 h, the liver showed a 53 times higher level of TNF-alpha and a 42 times higher level of IFN-gamma than the respective levels at the early time points after infection. The levels of TNF-alpha and IFN-gamma induced by LVS were 5 times lower than those induced by the Pohang isolate. Also, the organs from the Pohang-infected mice showed higher levels of TNF-alpha, IFN-gamma, IL-10 and IL-12 than the levels in the LVS-infected mice. The blood from the Pohang-infected mice at 120 h revealed about a 40 times increased level of IFN-gamma, and IL-10 was also increased by 4 times at 96 h compared to an early infection time point, while IL-4 was not induced during the whole infection period. These results suggest that F. tularensis may induce a Th1-mediated immune response to in vivo infection and the Pohang isolate has a higher capacity than the LVS to induce an acute immune response in Blab/c mice.

Diagnostic Experience in the 3 Human Brucellosis Cases by the Microbiologic, Serologic and Gene Tests / 대한임상미생물학회지

Brucellosis is a zoonosis caused by Brucella species. B. melitensis, B. suis, B. abortus and B. canis can infect humans. Recently, as the cases of bovine brucellosis have increased every year in Korea, the cases of human brucellosis have also increased among livestock workers and veterinarians in rural areas, since the first human case was reported in 2003. Because clinical manifestations of the disease are nonspecific and may be very atypical, clinicians and laboratory persons need to be active in using diagnostic tools including polymerase chain reaction in addition to the ordinary culture and serologic tests, and taking an appropriate measure to prevent intralaboratory infection. We report herein our experience in three human brucellosis cases diagnosed by cultures, serologic tests and gene detection.

Genotype Analysis of Pyrogenic Exotoxin and emm Genes of Streptococcus pyogenes Clinical Isolates

A variety of proteins produced by Streptococcus pyogenes contribute to the virulence of the pathogen. Among the proteins, the M protein and streptococcal pyrogenic exotoxins (Spe) are considered the major S. pyogenes virulence factors. To better characterize the correlation of M protein type and pyrogenic exotoxins with clinical diseases, we tested 269 S. pyogenes clinical isolates from patients with scarlet fever, pharyngitis, skin infection, otitis media, or other invasive streptococcal infections that provided appropriate clinical data. The strains were genotyped (M type) and assayed for speA, speB, and speC genes. The speB gene was detected in all isolates. Also, speA and speC genes were detected in 54 strains (18.2%) and 140 strains (47.3%), respectively. The strains isolated from invasive disease patients showed the highest frequency of speA gene (40.5%). The correlation among emm genotype, speA gene, and clinical patterns was analyzed. Genotypes emm1 (55.6%) and emm3 (22.2%) were predominant in stains with speA gene. The distribution of emm genotypes did not significantly associate with clinical patterns. These data suggest that SpeA is significantly associated with specific emm genotypes, and the exotoxin serve a dominant virulence factor.

Biochemical Characteristics and Antimicrobial Susceptibility Patterns of Vibrio vulnificus Isolated from the Environment of Korea in 2001

Vibrio vulnificus is a pathogen causing two types of severe illness, septicemia and wound infections, and is continually detected in marine environments. To investigate the biochemical characteristics and the antimicrobial susceptibility of V. vulnificus isolated from environment of Korea in 2001, the API 20E kit test, PCR, and antibiotic disk diffusion method were performed. A total of 210 V. vulnificus strains was isolated from seawater, shell-fish, sediments, coastal water, aquarium water, sewage, and others. All of the isolates could be divided into 15 groups on the basis of their API 20E profiles, and were positive in Indole test. Only 173 isolates (82.4%) were positive by the PCR amplifying the cytolysinhemolysin gene. Almost all isolates were susceptible to chloramphenicol (99.5%), tetracycline (90.0%), ciprofloxacin (92.4%), trimethoprim/sulfamethoxazole (89.0%), nalidixic acid (87.6%). Some isolates were resistant to cephalothin (57.6%), amikacin (33.3%), cefoxitin (31.9%). One hundred and forty three isolates (68.1%) were resistant to one or more antimicrobial agents. These results show that V. vulnificus environmental isolates possessed various biochemical characteristics, and some isolates were not detected of the cytolysin-hemolysin gene by PCR, and a part of isolates were resistant to multiple antibiotics.

Antigenic Analysis of Haemophilus influenzae Based on Biotypes and Patterns of OMP P2

No abstract available.

Genotypic and Phenotypic Analysis Among Clinical Isolate of Streptococcus pyogenes in Seoul , Korea

A total of 152 strains of Streptococcus pyogenes were isolated from patients with pharyngitis, scarlet fever, skin infection, or invasive streptococcal infections in Seoul, Korea from January 1988 to December 1999. All isolates were epidemiologically characterized to decide phenotypes by T protein serotype and serum opacity factor (OF) detection. Genetic diversity of the isolates were analyzed by emm genotyping and pulsed-field gel electrophoresis (PFGE). T protein serotype showed 17 kinds in distribution and T12 (40.1% of study strains), T4 (19.1%), and T1 (7.9%) were the prevalent ones. When sources of S. pyogenes isolates were analyzed by T serotype distribution, T12 type was predominant in pharyngitis and skin infection isolates which contributed to 30 strains (49.2%) and 11 strains (18.0%), respectively. When T serotype of S. pyogenes isolates were analyzed by emm genotype distribution, of the 61 isolates of T12 type, 48 strains (78.7%) belonged to the emm type 12 (M12) and of the 29 isolates of T4 type, 27 strains (93.1%) belonged to the emm genotype 4 (M4). PFGE of genomic DNA of different emm genotype (emm12, emm4 and emm1) showed distinctive patterns. When the DNA of same emm gene type isolates were analyzed genetic relatedness by PFGE pattern, emm4, emm1, and emm12 types showed over 90%, 75%, and 70% of genetic similarity, respectively. Therefore, it was suggested that these emm genotype isolates were closely related genetically whereas among the isolates of other emm genotypes showed less than 30% of genetic similarity. Show genotypes are more diverse in comparison with phenotypes. In even epidemiologically unrealated isolates, genetic subtypes appeared correlated. The phenotypic and genotypic analysis used in the study were discriminative and appropriate for epidemiological study of S. pyogenes.

Epidemiologic Characteristics of T Serotyping in Relation to the Outbreak of Erythromycin Resistant Streptocccus pyogenes

PURPOSE: The identification of antigenic specificity of Streptococcus pyogenes using T serotyping is important to understand biologic characteristics of microorganisrns. We would like to disover the association of the occurrence of predominant T type, with possible outbreak of erythromycin resistant Streptococcus pyogenes in this country, which has been documented since the late 1990s. METHODS: Throat swab cultures were taken from a total of 1,294 normal school children(Subject A) in two different geographical areas. A total of 92 strains(Subject B) were obtained from the patients with group A streptococcal infections from Jan. 1998 to Dec. 1998. All strains were serotyped with T protein antisera. RESULTS: The distribution of T12 in Uljin increased from 4.2%(1996) to 45.7%(1998). T4 increased from 6.3% to 20.0%. Thirty-eight out of 92 strains were resistant to erythromycin. Twenty-seven out of 41 strains(T12) were multidrug resistant to erythromycin, clindarnycin, and tetracycline. CONCLUSION: We can see the sudden increase in T12 strains, one of the strains that are resistant to erythromycin in 1998, compared with previous years. T protein serotyping could be epidemiologically useful as a screening methods for detecting erythromycin resistant group A streptococci in hospitals where the routine antibiotic sensitivity test dose not examin for streptococci.

Molecular Epidemiologic Study of Streptococcus pyogenes Analyzed by T protein Serotyping and Pulsed Field Gel Elecrophoresis(PFGE) in Normal Children

PURPOSE: In this study we tried to look at the spreading, duration of colonization, and acquisition of new streptococci which were obtained in one geographical area, as well as the bacteriologic and molecular epidemiology of normal school children carrying group A streptococci and their clonal relationship through the combined application of the serotype of T antigen and Pulsed Field Gel Electrophoresis(PFGE). METHODS: A total of 88 strains of group A streptococci were isolated from 396 normal school children. All isolates were classified in groups by Streptex and serotyped by T. agglutination. Restriction enzyme digestion of DNA was taken using Sma I. DNA fragments were separated by PFGE. RESULTS: A total of 33 strains were allocated their epidemiologic characteristics. Four out of 33 strains were not restricted by enzyme(Sma I). Twenty nine strains out of 33 strains showed 12 subtypes with 8-12 fragments between 40kbp and 500kbp of DNA fragments on PFGE. Eight strains of NT and T6 war same fragment patterns on PFGE, respectively. Three strains out of 4 strains of T8/25 were not restricted and the other one showed different, unique patterns. One strain out of 8 stains of T12 was not restricted, and the others were classified as 5 different subtypes. Two strains of Tl were different patterns from each other, and 2 strains of T4 showed the samefragment pattern CONCLUSION: T serotypes with PFGE will be useful as a screening and molecular epidemiologic method in a country where anti-M antisera is not available, after recognizing the advantages and disadvantages of M and T serotyping.

Pulsed field gel electrophoresis profile of erythromycin-clindamycin resistant Streptococcus pyogenes isolated in Korea

Ninety two strains of Streptococcus pyogenes were isolated from patients with pharyngitis, scarlet fever, skin infection, and invasive streptococcal infections in Seoul, Korea from January to December, 1998. All isolates were epidemiologically characterized by T protein serotype, and serum opacity factor (OF) detection to phenotypes. To analyze the genetic relationship, fifty two isolates including 32 erythromycin-clindamycin (Em-Cm) resistant strains, 20 antimicrobial susceptible strains were attempted to the pulsed-field gel electrophoresis (PFGE). T protein serotype showed 16 kinds in distribution including T12 and T4. Among the total isolates, 40 strains (43.5%) belonged to the T12 serotype and twenty strains (21.7%) to T4 serotype. On the other hand, when infection aspect of S. pyogenes isolates were analysed by T serotype distribution, T12 type was predominant for pharyngitidis which contributed to 21 strains (53%) and for skin infection isolates which contributed to 11 strains (28%), respectively. In case of T4 type, it was the most predominant pharyngitidis isolates which contributed to 8 strains (40%). In T serotype distribution of Em-Cm resistant strains, 27 strains (84%) of the thirty two showed T12 serotype. In minimum inhibitory concentration (MIC) values of Em-Cm resistance isolates, thirty two isolates showed resistant to erythromycin 27 strains (84%), had high MIC of >128 mug/ml. And also to clindamycin, twenty two strains (69%) had high MIC of >128 mug/ml. When OF detection of Em-Cm resistance of S. pyogenes isolates were analyzed by T serotype distribution, T12 serotype isolates revealed that all of the isolates except one strain were OF negative. In PFGE profile analysis to Em-Cm resistance isolates, of the twenty seven, Em-Cm resistance of T12 serotype isolates, 26 strains showed identical PFGE profile and all of these isolates revealed that OF negative. Eighty four percent of Em-Cm resistance S. pyogenes isolates had identical phenotype and PFGE profile. These results strongly suggested that the Em-Cm resistant S. pyogenes isolates from Seoul area showed close genetic correlation and PFGE could be available tool for molecular epidemiology.

Characterization of Haemophilus influenzae by SDS-PAGE, Restriction Enzyme Analysis and rRNA Gene Restriction Patterns

Among the fifty-three clinical isolates of Haemophilus influenzae, nineteen isolates including eight isolates of each biotype I-VIII, six of serotype b (Hib) strains and five of nontypeable strains were characterized by SDS-PAGE about outer membrane protein (OMP), restriction enzyme analysis (REA) and rRNA gene restriction pattems. OMP patterns showed to common band patterns in each H. influenzae isolate. Based on the two major proteins, 31KDa-38KDa, isolated strains were classified into 7 subtypes. In the OMP patterns about biotype and serotype, the specific pattern of each biotype was not distinguishable, but all of the serotype b strains were shown identical unique pattern, therefore it made distinctive difference with nontypeable strains. The digested genomic DNAs with EcoRI were identical result with rRNA gene restriction. It was more subdivided into 10 ribotypes. The most common ribotype I and serotype 1 accounted for 6 strains (31.6%) and 7 strains (36.8%) of the 19 clinical isolates, respectively. Hib isolates that were both OMP subtype 1 and ribotype I accounted for 2 strains (10.5%). In the epidemiologically unrelated strains, the putative association between the subtypes could not be confirmed. According to these results, the three methods were discriminatory and appropriate techniques for epidemiological studies of H. influenzae.

Retrospective survey of antibody response against borrelia burgdorferi in febrile pateints in 1990

No abstract available.