Pro-inflammatory Analysis of Macrophages in Contact with Titanium Particles and Porphyromonas gingivalis

Abstract During insertion of titanium dental implants, particles may shear from the implant to the periimplant region causing osteolysis, and their association with bacteria can exacerbate the inflammatory reaction. However, the association of a high invasive bacterium from the oral cavity, Porphyromonas gingivalis (Pg), and titanium particles remains unknown. This study evaluated pro-inflammatory reaction of human macrophages in contact with micro and nanoparticles of titanium associated with Porphyromonas gingivalis lipopolysaccharide (PgLPS). THP-1 cell were used and treated for 12, 24 and 48 h following 6 groups: Control(C), PgLPS (L); Microparticles (M); Nanoparticles (N); PgLPS and microparticles (LM); PgLPS and nanoparticles (LN). The following assays were carried out: i) cell viability using MTS, ii) cell morphology by SEM and iii) expression of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) by qRT-PCR and ELISA. For statistics two-way ANOVA followed by Tukey's test was used (p<0.05). After treatment, cells presented similar viability and morphology demonstrating that the treatments were not able to induce cell death. Gene expression was significantly higher for TNF-α and IL1-β after 12 h, and for IL-6 after 24 h in the N and LN groups. Cytokine production over time was an ascending curve for TNF-α with the peak at 48 h and IL1-β and IL-6 had a straight line among the time points, although cells from N group presented a significant production of IL-6 at 48 h. In conclusion, these results suggest that titanium nanoparticles stimulate stronger pro-inflammatory response in macrophages, independent of their association with LPS from P.gingivalis.

Resumo Durante a inserção de implantes dentários partículas de titânio podem ser liberadas na região peri-implantar levando ao processo de osteólise e a associação com a bactéria pode exacerbar ainda mais a reação inflamatória. Entretanto, a associação de uma bactéria altamente invasiva da cavidade oral, Porphyromonas gingivalis (Pg) e partículas de titânio ainda não foi investigada. Este estudo avaliou a reação pró-inflamatória de macrófagos humanos em contato com micro e nanopartículas de titânio associada a lipopolissacarídeo P. gingivalis (PgLPS). As células THP-1 foram utilizadas e tratadas durante 12, 24 e 48 h nos 6 seguintes grupos: Controle (C), PgLPS (L); micropartículas (M); nanopartículas (N); PgLPS e micropartículas (LM); PgLPS e nanopartículas (LN). Em seguida foram realizados os seguintes ensaios: i) a viabilidade celular utilizando MTS, ii) a morfologia celular por MEV e iii) expressão do fator de necrose tumoral alfa (TNF-α), interleucina-1 beta (IL-1β) e interleucina 6 (IL-6) por qRT-PCR e ELISA. Como estatística foi realizado o teste ANOVA two-way seguido pelo teste de Tukey (p<0,05). Após o tratamento, as células apresentaram viabilidade e morfologia semelhantes, demonstrando que os tratamentos não foram capazes de induzir a morte celular. A expressão de genes foi significativamente mais elevada para o TNF-α e IL1-β após 12h, e para a IL-6 após 24 horas em N e grupos de LN. A produção de citocinas em relação ao tempo representou uma curva ascendente para o TNF-α com o pico em 48 h, enquanto que para IL1-β e IL-6 se apresentou como uma linha reta com relação ao tempo, exceto pelo grupo N que foi significativo para IL-6 em 48 h . Conclui-se, a partir destes resultados, que as nanopartículas de titânio produziram o maior estímulo na resposta pró-inflamatória nos macrófagos, independente da sua associação com LPS de P. gingivalis.

Increased humoral antibody response of foot-and-mouth disease virus vaccine in growing pigs pre-treated with poly-γ-glutamic acid

This study was conducted to determine if humoral antibody response of foot-and-mouth disease (FMD) vaccine improved in 8-week-old growing pigs born to well-vaccinated sows pre-treated with 60 mg of poly-γ-glutamic acid (γ-PGA) three days before vaccination. Antibody against FMD virus serotype O was measured 0, 2, 4 and 6 weeks post-vaccination, using a PrioCHECK FMDV type O ELISA kit. The results showed that positive antibody reactions against FMDV serotype O antigen among a component of the vaccine significantly increased in response to pre-injection with γ-PGA.

Adherence and virulence genes of Escherichia coli from children diarrhoea in the Brazilian Amazon

The bacterial pathogen most commonly associated with endemic forms of childhood diarrhoea is Escherichia coli. Studies of epidemiological characteristics of HEp-2 cell-adherent E. coli in diarrhoeal disease are required, particularly in developing countries. The aim of this study was evaluate the presence and significance of adherent Escherichia coli from diarrhoeal disease in children. The prevalence of LA, AA, and DA adherence patterns were determined in HEp-2 cells, the presence of virulence genes and the presence of the O serogroups in samples obtained from 470 children with acute diarrhoea and 407 controls in Porto Velho, Rondônia, Brazil. E. coli isolates were identified by PCR specific for groups of adherent E. coli. Out of 1,156 isolates obtained, 128 (11.0%) were positive for eae genes corresponding to EPEC, however only 38 (29.6%) of these amplified bfpA gene. EAEC were isolated from 164 (14.1%) samples; of those 41(25%), 32 (19%) and 16 (9.7%) amplified eagg, aggA or aafA genes, respectively and aggA was significantly associated with diarrhoea (P = 0.00006). DAEC identified by their adhesion pattern and there were few isolates. In conclusion, EAEC was the main cause of diarrhoea in children, especially when the aggA gene was present, followed by EPEC and with a negligible presence of DAEC.

Characterization of Shiga-toxigenic Escherichia coli isolated from cases of diarrhoea & haemolytic uremic syndrome in north India.

Background & objectives: Shiga toxin producing Escherichia coli (STEC) is an important zoonotic foodborne pathogen, capable of causing haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS). As data from India on human infections caused by STEC are limited, this study was carried out for hospital based surveillance for STEC as a causative agent of diarrhoea, bloody diarrhoea and HUS at a tertiary care centre and to study the virulence gene profile and strain relatedness by multi locus variable tandem repeat analysis (MLVA). Methods: A total of 600 stool samples were studied. Stool samples of every fifth patient presenting with non-bloody diarrhoea, all cases of bloody diarrhoea and diarrhoea associated HUS (D+HUS) were collected from October 2009 to September 2011. Stool samples were cultured for STEC and characterization of STEC was done by serogrouping, virulence genes analysis, and MLVA typing. Results: STEC were isolated as a sole pathogen from 11 stool samples [5 of 290 (1.7%) non-blood diarrhoea and 5 of 300 (1.6%) blood diarrhoea cases]. STEC was also isolated from one fatal case of HUS who was an eight month old child. Only six of 11 isolates were positive for stx2 gene, whereas stx1 was present in all 11 isolates. Only one isolate was positive for eae. Other adhesion genes present were iha in five isolates, followed by toxB and efa1 in two each and saa gene in one, isolate. Among the plasmid encoded genes, espP, hly and etpD were each present in one isolate each. In the MLVA typing, diverse profiles were obtained except two untypeable isolates from different patients shared the same MLVA profile. Both these isolates were not epidemiologically linked. Interpretation & conclusions: This study demonstrated that STEC could be a causative agent of diarrhoea, bloody diarrhoea and sporadic HUS. However, further work needs to be done to study and explore the prevalence of these organisms in the food chain in this region.

Infección por Brucella canis en humanos: propuesta de un modelo teórico de infección a través de la ruta oral / Brucella canis infection in humans: Proposal of a theoretical model of infection through the oral route

La infección por Brucella canis en los humanos se ha reconocido recientemente como una zoonosis, pero frecuentemente es sub reportada debido a que los síntomas pueden confundirse con los de un resfriado común u otras infecciones causadas por otros patógenos. Los caninos son los hospederos primarios de Brucella canis ; el incremento en la tendencia de tener perros como mascotas podría también aumentar la posibilidad de transmisión de la infección a los humanos por el estrecho contacto entre la mascota infectada y su propietario. En Colombia, hay reportes de aislamientos de B. canis de caninos de criaderos y de un humano en contacto con perros infectados, al igual que reportes de caninos seropositivos a la infección. Sin embargo, no hay mucha información disponible sobre los mecanismos de interacción hospedero-patógeno que conduzcan al establecimiento de la infección por Brucella canis en perros y en humanos esta información es todavía menor. En esta revisión se propone un modelo para la infección humana con Brucella canis a través de la ruta oral utilizando la información disponible para otras especies de Brucella que infectan al humano, incluyendo B. abortu s y B. melitensis , que difieren de B. canis en la composición estructural de su lipopolisacárido. También se hipotetiza el mecanismo de infección celular que es usado por B. canis para invadir y establecer la infección en células no fagocíticas y fagocíticas.

Brucella canis infection in humans has recently been recognized as a zoonosis, but it is frequently under reported because the flu-like symptoms are often confused with the presence of other disease-causing pathogens. Dogs are the primary hosts for Brucella canis ; the increasing trend to adopt dogs as pets also enhances the likelihood of transmission of Brucella canis infection through contact between infected dogs and owners. In Colombia, there are reports of isolates of B. canis from kennel dogs and also from one human being along with seropositive results from dogs and humans. However, the mechanism of hostpathogen interactions leading to the infection of Brucella canis in dogs is still unknown and even less is known about human infections. This review proposes a model for human infection with Brucella canis through the oral route. We use the information available for other human-infecting Brucella species, including B. abortu s and B. melitensis, which differ from B. canis in the structural composition of the lipopolysaccharide molecule. The mechanism of cellular infection used by B. canis to invade and establish infection in nonphagocytic and phagocytic cells is also hypothesized.

The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

Shigella flexneri causes bacillary dysentery in humans. Essential to the establishment of the disease is the invasion of the colonic epithelial cells. Here we investigated the role of the lipopolysaccharide (LPS) O antigen in the ability of S. flexneri to adhere to and invade polarized Caco-2 cells. The S. flexneri 2a O antigen has two preferred chain lengths: a short O antigen (S-OAg) regulated by the WzzB protein and a very long O antigen (VL-OAg) regulated by Wzz pHS2. Mutants with defined deletions of the genes required for O-antigen assembly and polymerization were constructed and assayed for their abilities to adhere to and enter cultured epithelial cells. The results show that both VL- and S-OAg are required for invasion through the basolateral cell membrane. In contrast, the absence of O antigen does not impair adhesion. Purified LPS does not act as a competitor for the invasion of Caco-2 cells by the wild-type strain, suggesting that LPS is not directly involved in the internalization process by epithelial cells.

Comparison of multiplex PCR with serogrouping and PCR-RFLP of fliC gene for the detection of enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) comprise one of the six categories of diarrhoeagenic E. coli (DEC). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC). The identification of DEC cannot be based only on cultural and biochemical criteria, since they are indistinguishable from the non-pathogenic E. coli commonly found in human feces. Several PCR methods, with both single and multiple target genes, have been reported for detecting the different DEC pathotypes. In the present study five hundred E. coli isolates from children with diarrhea were subjected into multiplex PCR. Furthermore the strains were typed serologically with O antisera and their fliC gene was characterized by PCR-RFLP. The results obtained revealed that overall 41 (8.2 percent) isolates could be detected as EPEC by this multiplex PCR assay. Of these isolates; 27 (66 percent) were typical (escv+, bfp+) and 14 (34 percent) atypical EPEC (escv+, bfp-). None of these 41 isolates contained the Stx1 and Stx2 genes. Among 37 (90 percent) typeable strains, nine different serogroups were present. The most common serogroups were O111, followed by O86, O55 and O119 and 10 different H types were found among these isolates. The multiplex PCR assay was found to be rapid and reliable in comparison to serological test; especially when screening the large number of isolates.

Relationship between O serotype and virulent genes in Escherichia coli causing urinary tract infections

Escherichia coli are the most frequent pathogens in acute urinary infections. They are classified based on various types of O antigen. Escherichia coli strains that cause urinary tract infections possess several genes encoding urovirulent factors. To assay the relation of virulent factors of E coli in acute urinary infections, the serotypes and virulence factor genotypes were determined. We studied 96 E coli isolates from children with acute urinary infections. Four urovirulence determinants were analyzed by DNA colony hybridization, including the genes for type 1 fimbriae [pil], P fimbriae [pap], S fimbriae [sfa], hemolysin [hly], and cytotoxic necrotizing factor 1 [cnfl]. O serotypes were also determined. The most frequently found virulence factor-encoding gene in the E coli strains studied was the gene for type 1 fimbriae [27.4%]. The prevalence of pap, sfa, hly, and cnf1 were higher in serotypes causing pyelonephritis than cystitis. The most common type of O antigen was O1 [12.2%]. There was a significant correlation between serotype and genotype in uropathogenic E coli. The high prevalence of O6 serotypes in children urinary tract infections and the high percentage of virulent genes in serotype O6 suggested a close relation between serotype and genotypes of uropathogen E coli

Multiplicación de Brucella abortus y producción de óxido nítrico en dos líneas celulares de macrófagos de distinto origen / Multiplication of Brucella abortus and production of nitric oxide in two macrophage cell lines of different origin

Brucella abortus es una bacteria que causa abortos e infertilidad en el ganado y fiebre ondulante en el hombre. Se multiplica en el citoplasma celular evadiendo los mecanismos de muerte intracelular. El óxido nítrico (NO) es importante en la regulación de la respuesta inmune. En el presente trabajo estudiamos la habilidad de tres cepas de B. abortus para sobrevivir intracelularmente en dos líneas celulares de macrófagos. La multiplicación de bacterias en ambas líneas celulares fue determinada a distintos tiempos en número de UFC/ml, también fue observada al microscopio de campo claro y de fluorescencia utilizando Giemsa y naranja de acridina, respectivamente. La tinción de ambas líneas celulares inoculadas con B. abortus mostró un resultado concordante con el encontrado en la determinación del número de UFC. Fue confirmada la presencia de B. abortus por microscopía electrónica. Para medir la producción de NO se utilizó el reactivo de Griess. La multiplicación de la cepa rugosa RB51 disminuyó en ambas líneas celulares y los niveles de NO fueron mayores en células inoculadas con dicha cepa que cuando fueron inoculadas con las cepas lisas (S19 y 2308). Estos resultados sugieren que probablemente la ausencia de cadena O en el lipopolisacárido afecta el crecimiento intracelular de B. abortus.

Brucella abortus is a bacterium which causes abortions and infertility in cattle and undulant fever in humans. It multiplies intracellularly, evading the mechanisms of cellular death. Nitric oxide (NO) is important in the regulation of the immune response. In the present work, we studied the ability of three B. abortus strains to survive intracellularly in two macrophage cell lines. The bacterial multiplication in both cell lines was determined at two different times in UFC/ ml units. Moreover the inoculated cells were also observed under light-field and fluorescence microscopy stained with Giemsa and acridine orange, respectively. The stain of both cellular lines showed similar results with respect to the UFC/ml determination. The presence of B. abortus was confirmed by electronic microscopy. In both macrophage cell lines inoculated with RB51, the multiplication diminished and the level of NO was higher, compared with cells inoculated with smooth strains (S19 and 2308). These results suggest that the absence of O-chain of LPS probably has affects the intracellular growth of B. abortus.

Antigenic competition among different 'O' antigens of Salmonella enterica subspecies enterica serovars during hyperimmunization in pony mares.

The present study on antigenic competition among somatic 'O' antigens of different Salmonella groups (A, B, C1, C2, D and E1) in mares revealed that the immune response to most of the antigens was not (A, B, C2) or little (C1, D) affected by antigenic competition. However, E1 group antigen, which induced high antibody titres (Avg. 12967.3) when given alone, produced almost 3.5 log2 lower antibody titres on giving with other antigens, indicating the antigenic competition among some Salmonella group antigens. The antigenic competition varied for different antigens even of the similar chemical nature. Therefore, antigens belonging to different somatic groups should not be given together for the purpose of raising polyvalent serum or for immunization using multivalent Salmonella vaccines prepared from strains of different 'O' groups revealing antigenic competition.

Changes of serum neuron specific enolase in rats with septic shock / 中华儿科杂志

<p><b>OBJECTIVE</b>To study the changes of serum neuron specific enolase in rats with septic shock.</p><p><b>METHODS</b>The model of septic shock was set up by injection of lipopolysaccharide (LPS, from Escherichia coil O55: B5) at a dose of 25 mg/kg through femoral vein. Twenty male Wistar rats were randomly divided into 2 groups: normal control group (LPS was substituted by same volume of normal saline solution) and septic shock group. Six hours after the septic shock model formed, whole blood was taken for measuring the serum neuron specific enolase (NSE). The brains of the rats were taken for histopathological examination.</p><p><b>RESULTS</b>The serum NSE of septic shock group was significantly higher than that of control group [(10.0781 +/- 0.526) microg/L vs. (3.7188 +/- 0.602) microg/L, P < 0.05]. Neurons were severely damaged 6 hours after injection of LPS. Neuronal necrosis and the damage of blood-brain barrier were seen by light and electron microscope in septic shock group but not in the control group.</p><p><b>CONCLUSION</b>NSE in serum increased when septic encephalopathy occurred, which indicated that NSE might become a marker of neural damage in septic shock.</p>

Serum resistance of Escherichia coli strains causing urinary tract infection and diarrhoea in relation to alpha haemolysin production and O type.

A total of 46 alpha-hemolytic and 40 non-hemolytic clinical isolates of Escherichia coli were collected from pediatric patients with urinary tract infection and diarrhoea. Of 39 (84.7%) alpha-hemolytic strains and 27 (67.5%) non-hemolytic strains were resistant to 10% serum and there was no significant difference between urinary and stool isolates. On the contrary when 100% serum was used, 22 (47.8%) of the alpha-hemolytic and 7 (17.5%) of the non-hemolytic strains were resistant (p<0.01). and significantly greater resistance was found in urinary tract infection than from the stool samples (47% versus 24%, p<0.01). Serum resistance was higher in serogroups O6, O18 and O75. Production of alpha-hemolysin was more frequent in serogrops O2, O6, O8, O18 and O75. Thus, the resistance to human serum can determine clinical significance of Escherichia coli from different sources and alpha-hemolysin contributes to the virulence of Escherichia coli in initiation and perpetuation of clinical infection.

A Molecular Epidemiologic Study by Plasmid DNA and Random Amplified Polymorphic DNA Analyses of Salmonella enterica serotype Enteritidis Isolated in Food Borne Outbreaks in Gwangju, 1997

BACKGROUND: During the past 10 years, there has been an increased incidence of gastrointestinal infections caused by salmonellae in Korea. In 1999, there were several outbreaks and sporadic occurrences of food borne infections due to Salmonella enterica serotype Enteritidis in Gwangju. Thus, there is a need for careful monitoring of its occurrence. METHODS: Salmonella Enteritidis were isolated from feces samples of patients with foodborne diarrhea in Gwangju, 1999. We performed antigen typing, examination of biochemical properties, anibiotic susceptibility test, plasmid typing and RAPD analysis to characterize of S. Enteritidis isolates. RESULTS: There were three Salmonella outbreaks (April, July, October), and 203 isolates of S. Enteritidis were isolated from the 286 patients. Eighteen isolates were obtained from the patients of sporadic occurrences. Antigenic types of the isolates were O antigen; D1 (1, 9, 12), H antigen phase 1; (g, m), serotype; Enteritidis. The isolates were susceptible to most of the antibiotics. We performed plasmid DNA analysis of the isolates, and the results showed 3 plasmids (8, 6, 3.8 kb) in 14 of 14 strains from outbreaks; 3 plasmids (8, 6, 3.8 kb) in 2 isolates from sporadic cases; 4 plasmids (8, 6, 3.8, 2 kb) in 10 isolates from sporadic occurrences, and 2 isolates from food specimens. However, 1 isolate from patients and 2 isolates from Ham-Yang, Kyung Nam, did not contain plasmids. RAPD analysis showed that all isolates from Gwangju in 1999 showed relatively uniform characteristics which were different from those derived from Ham-Yang, Kyung-Nam. CONCLUSION: The present data demonstrated that most food poisoning cases by S. Enteritidis in Gwangju, 1999, were originated from the same Salmonella Enteritidis strains.

A Molecular Epidemiologic Study by Plasmid DNA and Random Amplified Polymorphic DNA Analyses of Salmonella enterica serotype Enteritidis Isolated in Food Borne Outbreaks in Gwangju, 1997

BACKGROUND: During the past 10 years, there has been an increased incidence of gastrointestinal infections caused by salmonellae in Korea. In 1999, there were several outbreaks and sporadic occurrences of food borne infections due to Salmonella enterica serotype Enteritidis in Gwangju. Thus, there is a need for careful monitoring of its occurrence. METHODS: Salmonella Enteritidis were isolated from feces samples of patients with foodborne diarrhea in Gwangju, 1999. We performed antigen typing, examination of biochemical properties, anibiotic susceptibility test, plasmid typing and RAPD analysis to characterize of S. Enteritidis isolates. RESULTS: There were three Salmonella outbreaks (April, July, October), and 203 isolates of S. Enteritidis were isolated from the 286 patients. Eighteen isolates were obtained from the patients of sporadic occurrences. Antigenic types of the isolates were O antigen; D1 (1, 9, 12), H antigen phase 1; (g, m), serotype; Enteritidis. The isolates were susceptible to most of the antibiotics. We performed plasmid DNA analysis of the isolates, and the results showed 3 plasmids (8, 6, 3.8 kb) in 14 of 14 strains from outbreaks; 3 plasmids (8, 6, 3.8 kb) in 2 isolates from sporadic cases; 4 plasmids (8, 6, 3.8, 2 kb) in 10 isolates from sporadic occurrences, and 2 isolates from food specimens. However, 1 isolate from patients and 2 isolates from Ham-Yang, Kyung Nam, did not contain plasmids. RAPD analysis showed that all isolates from Gwangju in 1999 showed relatively uniform characteristics which were different from those derived from Ham-Yang, Kyung-Nam. CONCLUSION: The present data demonstrated that most food poisoning cases by S. Enteritidis in Gwangju, 1999, were originated from the same Salmonella Enteritidis strains.

Novel Vaccine Development against Escherichia coli O157: Immunologic Properties of O-Specific Polysaccharide-Shiga-like Toxin Conjugate Vaccines

Escherichia coli (E. coli) O157:H7 is an important cause of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). LPS-based vaccines need improvement since the anti-LPS antibodies raised by the vaccines are bactericidal and release toxin that may precipitate the development of HUS. Despite huge efforts, the treatment of infection with E. coli O157 has been difficult because antibiotics do not change the course of the enteritis caused by E. coli O157 and may increase the incidence of HUS through the release of Shiga-like toxin (Stx)-I. For this aim we tried the conjugate of E. coli O157 OSP bound to the nontoxic B subunit of Stx-I B as a vaccine that can induce both serum IgG anti-LPS antibody with bactericidal activity and neutralizing antibody to Stx-I. Mice were immunized s.c. with OSP-Stx-I B conjugate. Anti-sera were analyzed for the anti-LPS antibody, anti-Stx-I B antibody, complement-dependent bactericidal activity, and Stx-I neutralization activity. Mice injected with the bivalent conjugate elicited both bactericidal antibodies to E. coli O157 and neutralization antibodies to Stx-1. We also analyzed the distributional, functional changes of T lymphocytes in vitro and in vivo. After the injection of Stx-I, splenocytes showed a decrease in proliferation when stimulated with phytohemagglutinin (PHA) or LPS, and the number of CD4+ and CD8+ T cells also decreased. Interleukin (IL)-2, IL-4 and IFN-gamma productions by CD3+ T cells were slightly increased in the Stx-I injected mice.

Principales características y diagnóstico de los grupos patógenos de Escherichia coli / Principal characteristics and diagnosis of the pathogenic groups of Escherichia coli

Escherichia coli colonizes the human intestinal tract within hours of birth and is considered a non-pathogenic member of the normal intestinal flora. However, there are six pathogenic groups that may produce diarrhea: enterotoxigenic (ETEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC), enteroaggregative (EAEC) and diffusely adherent (DAEC) groups. E. coli can be isolated and classified using traditional methods, by identifying its biochemical or serum characteristics. The pathogenic mechanisms may be studied in cell cultures and animal model assays, as well as more up to date molecular biology methods for study and diagnosis. The latter have proven that genes are involved in pathogenesis. The objective of the present work is to draw attention to the importance of E. coli as a pathogenic organism. This microorganism is an etiologic agent of sporadic cases of diarrhea, hemorrhagic colitis, dysentery, and hemolytic uremic syndromes and outbreaks. Diarrheic E. coli manifestations occur mainly among infants, and deep knowledge and understanding of this microorganism are crucial to better epidemiologic surveillance.

Development of Test System for Detection of Antibody to Human Immunodeficiency Virus Type 1 Subtype O

In Korea, all domestic made test systems for detecting antibodies in HIV-1 contain the antigens from human immunodeficiency type 1 (HIV-1) subtype B. However, because HIV-1 subtype O is significantly different in amino acid sequences from all other subtypes of HIV-1, there has been a need for developing a test for detecting antibodies in subtype O. For this purpose, the entire nucleotide sequence corresponding to the extracellular domain of the transmembrane glycoprotein of HIV-1 subtype O was synthesized with consideration of Escherichia coli cordon usage. Various regions of the extracellular domain were cloned into E. coli expression vectors and tested for levels of protein production. The nucleotide sequence, named ECTM, that can encode a 129 amino acid-long peptide, was found to be expressed at a high level in E. coli. The protein of approximately 17 kDa specifically reacted with sera from individuals infected with HIV-1 subtype O. The ECTM protein was purified to near homogeneity by the CM-T gel chromatography, using concentrated, denatured inclusion bodies. In Western blot analysis, the purified viral antigen reacted with sera from individuals infected with subtype O more efficiently than subtype B. The enzyme linked immunoabsorbent assay (ELISA) system was developed using the subtype O viral protein and compared with the commercially available kit lacking the antigens from subtype O. The ELISA kit containing the subtype O antigen ECTM alone efficiently reacted with sera from individuals infected with subtype O. The subtype O antigen-containing kit produced a positive absorbence even when sera were diluted 512-fold, suggesting a high sensitivity. The commercially available kit also reacted with subtype O sera, but produced a negative result at a dilution of 8-fold. Our results suggest that the currently available kit may not be able to efficiently detect subtype O sera and that the viral protein developed in this study may be added to the current system to maximize the detection of sera from individuals infected with subtype O.

100 years of Widal test & its reappraisal in an endemic area.

A reappraisal of the Widal test was made for its diagnostic utility in typhoid fever in an endemic area of Central India. The significant basal antibody level in the normal population based on 1200 voluntary/relative blood donors at the cut-off titre of 80 or above was observed in 13.83 and 8.0 per cent for 'O' and 'H' antigens of Salmonella typhi respectively. A retrospective study (1991-1995) over 138 bacteriologically proven cases of typhoid showed a positivity of 64.49 and 78.26 per cent respectively for 'O' and 'H' antibodies at the titre of 80 or above and 44.2 and 63.04 per cent at the titre of 160 and above. The retrospective data also showed a greater positivity (46.41%) in 1991 which decreased to 25 per cent in 1995 and appeared to follow the incidence of multi drug resistant S. typhi over the period. The detection of 'H' antibodies is no less important than the 'O' antibodies in the present study. Our data bring out the diagnostic limitations of Widal test done on single samples collected in the early phase of illness (4-10 days) from patients suspected to have typhoid in an endemic area of Central India.

Epidemiological Investigation of Yersinia pseudotuberculosis Infection in Korean / 대한임상병리학회지

BACKGROUND: The purpose of this study is to prove that human infection of Yersinia pseudotuberculosis might be occurred by drinking of mountain spring water contaminated with wild mice excreta through epidemiological tool. METHOD: Y. pseudotuberculosis strains which were isolated from patient stools, mountain spring water and mice excreta were analysed by serotyping of O antigen and plasmid DNA profile (Restriction Endonuclease Analysis of Plasmic DNA analysis REAP) assay Also reservoir rate of Y. pseudotuberculosis was calculated from wild mice which were captured throughout Korean mountains. RESULTS: Reservoir rate of Y. pseudotuberculosis from wild mice in Korea was 0.85% and was not higher than that in other country. The analysis of 66 strains of Y. pseudotuberculosis showed that 36 strains of serotype 15, REAP B type, 24 strains of serotype 4b, REAP D type, and 1 strain of serotype 4b, REAP new unclassifiable type, but 5 strains didn't have plasmic (serotype 15:3, 11 :2) .Especially same 4b, D type of Y. pseudotuberculosis was isolated from patient stools, mountain spring water and wild mouse (Apodemus agrarius) excretion and this fact was considered that Y. pseudotuberculosis strains from 3 groups were closely correlated epidemiologically. Also serotype 15, REAP B strains were isolated from patient stools and mountain spring water, but were not isolated from wild mice yet and 15, B type was isolated from Korea only and considered as native Korean strain which had not isolated in other countries yet. CONCLUSIONS: Human Y. pseudotuberculosis infection in Korea was occurred by drinking of contaminated mountain spring water and A. agrarius was one of main reservoir which contaminates mountain spring waters in Korea, Also above antigenic distribution of Y. pseudotuberculosis would be useful for development of ELISA kit of Korean type.