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PubMed1961

Labeling procedures employing crystalline fluorescein isothiocyanate.

Griffin, Charles W. (Baltimore Biological Laboratory, Baltimore, Md.), Theodore R. Carski, and George S. Warner. Labeling procedures employing crystalline fluorescein isothiocyanate. J. Bacteriol. 82:534-537. 1961.-The effect of altering the ratio of dye to protein in the labeling of three immune globulins with crystalline fluorescein isothiocyanate was the purpose of the study. Direct staining of group A streptococci and rabies-infected mouse brain, and indirect staining of Treponema pallidum (Nichols strain) comprised the three immunological systems selected for investigation. Maximal specific staining and minimal nonspecific background staining were demonstrated by the use of low dye-to-protein labeling ratios.

Animals↗
PubMedDate not supplied

Prospects for improved laboratory diagnoses of treponemal infections and species differentiation.

The serologic diagnosis of treponemal infections has depended in the past on a variety of tests in which specificity was defined on an epidemiologic rather than on an immunologic basis. The lipoidal antigen tests possess no immunologic specificity. Tests based on whole treponemal antigens, although they do have some immunologic specificity, react with antibodies other then those generated in the course of syphilis and yaws infections. Recent developments in biotechnology now permit the identification of immunologically specific antigens in Treponema pallidum, and cloning of appropriate genetic information in Escherichia coli has led to the production of pure specific reagents. These developments will finally place the serologic diagnosis of treponemal infections on a sound immunologic basis.

Animals↗
PubMed2000

Risk factors for HTLV-I and II in individuals attending a clinic for sexually transmitted diseases.

BACKGROUND: To date, few studies have provided information on risk factors for human t-lymphotropic viruses (HTLV) types I and II in European countries. In particular, few data are available from published studies conducted in STD centers. GOALS: To identify risk factors for HTLV-I and HTLV-II infection and to better distinguish the epidemiologic patterns of the two viruses in Italy. STUDY DESIGN: A cross-sectional study of individuals at high risk of sexually or parenterally transmitted infections attending a large STD center in an urban setting was conducted. Serologic tests for HTLV-I and II, HIV, hepatitis virus type B (HBV), hepatitis virus type C (HCV), and syphilis were performed. Information regarding at-risk behavior was collected using a specific questionnaire. RESULTS: From January 1994 to June 1996, 1,457 individuals were recruited; of them, 1,016 (69.7%) were males, 1,051 (72.4%) Italians, and 288 (19.8%) non-Europeans. One thousand seventy-five (74.8%) participants were noninjecting-drug-using heterosexuals, 285 (19.6%) were men who have sex with men, and 97 (6.6%) were injecting drug users (IDU). The mean age of the study participants was 33.6 (+/-10.5) years. Nine (0.6%) individuals were positive for HTLV-I antibodies and 9 (0.6%) for HTLV-II antibodies. The prevalence of HTLV-I among IDUs, men who have sex with men, and noninjecting-drug-using heterosexuals, was 2.1% (2/97), 1.4% (4/ 285), and 0.3% (3/1085), respectively. HTLV-II prevalence was 8.2% (8/97) among IDUs and 0.09% (1/1075) among noninjecting-drug-using heterosexuals. Among the nine HTLV-II-positive individuals, eight were Italian IDUs and one was a noninjecting-drug-using heterosexual man from India. None of the 285 men who have sex with men had HTLV-II antibodies. HTLV-infected individuals tended to be older than those who were uninfected. HTLV-I-infected individuals were more likely to be non-European and to have antibodies against Treponema pallidum. Injecting drug use tended to be independently associated with HTLV-II infection. CONCLUSIONS: The data suggest a role of sexual behavior in the spread of HTLV-I, which is more likely to be detected in individuals coming from endemic areas. Injecting drug use remains the most important risk factor for HTLV-II infection in Italy.

Adolescent↗
PubMed2003

PCR methodology as a valuable tool for identification of endodontic pathogens.

OBJECTIVES: This paper reviews the principles of polymerase chain reaction (PCR) methodology, its application in identification of endodontic pathogens and the perspectives regarding the knowledge to be reached with the use of this highly sensitive, specific and accurate methodology as a microbial identification test. DATA SOURCES: Studies published in the medical, dental and biological literature. STUDY SELECTION: Evaluation of published epidemiological studies examining the endodontic microbiota through PCR methodology. CONCLUSIONS: PCR technology has enabled the detection of bacterial species that are difficult or even impossible to culture as well as cultivable bacterial strains showing a phenotypically divergent or convergent behaviour. Moreover, PCR is more rapid, much more sensitive, and more accurate when compared with culture. Its use in endodontics to investigate the microbiota associated with infected root canals has expanded the knowledge on the bacteria involved in the pathogenesis of periradicular diseases. For instance, Tannerella forsythensis (formerly Bacteroides forsythus), Treponema denticola, other Treponema species, Dialister pneumosintes, and Prevotella tannerae were detected in infected root canals for the first time and in high prevalence when using PCR analysis. The diversity of endodontic microbiota has been demonstrated by studies using PCR amplification, cloning and sequencing of the PCR products. Moreover, other fastidious bacterial species, such as Porphyromonas endodontalis, Porphyromonas gingivalis and some Eubacterium spp., have been reported in endodontic infections at a higher prevalence than those reported by culture procedures.

Bacteria↗
PubMed1995

Polymerase chain reaction for identification of human and porcine spirochaetes recovered from cases of intestinal spirochaetosis.

A polymerase chain reaction (PCR) amplification of 16S rDNA was developed to identify spirochaetes recovered from cases of intestinal spirochaetosis in humans and pigs; these bacteria belong to a distinct genetic group of spirochaetes, with the proposed name 'Anguillina coli'. The PCR incorporated a universal eubacterial 16S rDNA sequencing primer (1492r), and a 21-base forward primer designed to include a nucleotide sequence specific for 'A. coli'. The PCR was used to correctly identify DNA extracted from 43 isolates of 'A. coli' from humans and pigs, whilst no product was produced from Escherichia coli, or from other intestinal spirochaetes, including 38 isolates of Serpulina spp., and one each of Treponema succinifaciens and Brachyspira aalborgi. The amplification provided a rapid and simple means of identifying DNA from isolates of 'A. coli', and could be used on boiled whole 'A. coli' cells, with a detection limit equivalent to 2.5 x 10(2) cells. The reaction was used to detect and identify these spirochaetes from selective agar plates inoculated with stool specimens from infected pigs.

Animals↗
PubMed1990

Development of a diagnostic test for anaerobic periodontal infections based on plaque hydrolysis of benzoyl-DL-arginine-naphthylamide.

Treponema denticola, Porphyromonas (Bacteroides) gingivalis, and Bacteroides forsythus are among the anaerobic species frequently associated with adult forms of periodontal disease. These organisms hydrolyze the synthetic peptide benzoyl-DL-arginine-naphthylamide (BANA), and such enzyme activity can be detected in the plaque and related to clinical disease and the presence of spirochetes. In this investigation, the liquid BANA assay was compared with a commercially developed BANA assay which employed a paper format and which could be read after a 15-min incubation. In the paper format, strips of a Whatman filter paper were impregnated with BANA and strips of nitrocellulose paper were impregnated with fast black K salt. Both strips were applied lengthwise across a paper card (3 by 5 in. [7.6 by 12.7 cm]). The BANA strip at the bottom was inoculated with the test sample (pure culture, plaque), folded back so that it contacted the fast black strip, and then incubated for 15 min at 55 degrees C. T. denticola, P. gingivalis, and B. forsythus always gave a positive reaction, whereas 51 other plaque species were always negative. Six Bacteroides and Capnocytophaga species on occasion had weak reactions. The proportional agreement between BANA positiveness and clinical disease was similar for both the liquid and the paper assays. The sensitivity, specificity, and accuracy relative to the clinical standard of the liquid assay were 74, 76, and 77%, respectively, while those of the paper assay were 81, 78, and 80%, respectively. The paper assay was significantly associated with the presence of either T. denticola or P. gingivalis or both in the plaque samples, with a sensitivity of 85%, a specificity of 53%, and an accuracy of 79%. These findings indicate that a rapid paper assay for BANA hydrolysis gives data comparable to those obtained with the liquid BANA assay.

Arginine↗
PubMed1997

Conservation of the 15-kilodalton lipoprotein among Treponema pallidum subspecies and strains and other pathogenic treponemes: genetic and antigenic analyses.

The 15-kDa lipoprotein of Treponema pallidum is a major immunogen during natural syphilis infection in humans and experimental infection in other hosts. The humoral and cellular immune responses to this molecule appear late in infection as resistance to reinfection is developing. One therefore might hypothesize that this antigen is important for protective immunity. This possibility is explored by using both genetic and antigenic approaches. Limited or no cross-protection has been demonstrated between the T. pallidum subspecies and strains or between Treponema species. We therefore hypothesized that if the 15-kDa antigen was of major importance in protective immunity, it might be a likely site of antigenic diversity. To explore this possibility, the sequences of the open reading frames of the 15-kDa gene have been determined for Treponema pallidum subsp. pallidum (Nichols and Bal-3 strains), T. pallidum subsp. pertenue (Gauthier strain), T. pallidum subsp. endemicum (Bosnia strain), Treponema paraluiscuniculi (Cuniculi A, H, and K strains), and a little-characterized simian isolate of Treponema sp. (Fribourg-Blanc strain). No significant differences in DNA sequences of the genes for the coding region of the 15-kDa antigen were found among the different species and subspecies studied. In addition, all organisms showed expression of the 15-kDa antigen as determined by monoclonal antibody staining. The role of the 15-kDa antigen in protection against homologous infection with T. pallidum subsp. pallidum Nichols was examined in rabbits immunized with a purified recombinant 15-kDa fusion protein. No alteration in chancre development was observed in immunized, compared to unimmunized, rabbits, and the antisera induced by the immunization failed to enhance phagocytosis of T. pallidum subsp. pallidum by macrophages in vitro. These results do not support a major role for this antigen in protection against syphilis infection.

Animals↗
PubMed2004

Immunophenotypes, apoptosis, and expression of Fas and Bcl-2 from peripheral blood lymphocytes in patients with secondary early syphilis.

OBJECTIVES: Whether syphilis influences lymphocyte apoptosis has not been reported so far. GOAL: The goals of this study were to determine whether syphilis influences lymphocyte apoptosis and to investigate the molecular mechanism of apoptosis. STUDY DESIGN: Peripheral blood lymphocyte (PBL) immunophenotypes, apoptosis, and expression of Fas (CD95) and Bcl-2 were detected by flow cytometry in 33 patients with secondary early syphilis and 30 healthy subjects. RESULTS: Compared with the control subjects, the percentage of CD4 T cells and the ratio of CD4:CD8 were significantly decreased, and that of CD8 T cells and the apoptotic rates of PBLs and CD4 T cells were obviously increased in syphilitic patients. Fas overexpression and Bcl-2 downexpression in the syphilitic group were observed in PBLs and CD4 cells but not in CD8 and CD19 cells. CONCLUSIONS: The increased apoptosis of PBLs and CD4 T cells by Fas-mediated death pathway and downexpression of Bcl-2 protein could account for immune dysfunction in secondary early syphilis, which is responsible for the incomplete clearance of Treponema pallidum from the lesions and the chronic infection.

Adolescent↗
PubMed1980

Characterization of lymphocyte responsiveness in early experimental syphilis. I. In vitro response to mitogens and Treponema pallidum antigens.

Lymphoid cells from spleens and lymph nodes of rabbits infected with T. pallidum respond by proliferation to concanavalin A (Con A) and T. pallidum antigens. Spleen cell responsiveness to treponemal antigens appears 6 days after infection, is 100 to 600 fold higher than the response of uninfected control rabbits, and is maintained throughout the 31-day observation period. Specifically responding cells in the inguinal and popliteal lymph nodes of infected animals are demonstrable on day 10, and the magnitude of the response increases throughout the observation period. Specific responsiveness to T. pallidum antigens in vitro is enhanced in purified T cell populations and is abolished by treatment with goat anti-rabbit thymocyte serum and complement. The response of spleen and lymph node cells to Con A is unaffected during syphilitic infection. These results are consistent with a role for T cell-mediated specific immunity to treponemal antigens early after infection and do not support a hypothesis of depressed cellular immunity during syphilitic infection.

Animals↗
PubMed1996

The peptidolytic capacity of the spirochete system.

Relatively scant chemical information has been available on the proteinases and peptidases of spirochetes in spite of the association of spirochetes with several serious infections known to plague humans and other animal species. This situation has partly resulted from difficulties in growing some spirochetes under laboratory conditions. The cells of Treponema denticola, a spirochete suggested to be associated with periodontal infections, have turned out to be a good source of new chemical information on those enzymes. Latest studies suggest that the outer cell envelope or the periplasmic space of T. denticola contains several novel proteinases and peptidases (hence called "ectoenzymes") which may contribute to the chronicity of periodontal infections. Some of the oligopeptidases discovered are specific for proline-containing host tissue peptides such as substance P, bradykinin, neurotensin, etc., and possibly small collagen fragments. The only spirochetal peptidases purified to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been obtained from T. denticola. One particular peptidase, suggested to be similar to the oligopeptidase B (EC 3.4.21.83) of Escherichia coli seems to be present in the cell envelope or in the periplasmic space at quite large concentrations. The presence of this and several other peptidases in the outer cell structures of the treponemes suggests that such enzymes are important for the nutrition of these highly motile and invasive organisms. The biological role of these enzymes can thus be envisaged in the peptidolytic processing of host tissue proteins and peptides to gradually smaller molecules to fulfill the nutritional requirements of these organisms. Although the genetic similarity between T. denticola and some other treponemes and spirochetes can be hotly debated, it is nevertheless now possible to use T. denticula enzymes as suitable objects for comparison when the chemistry of other spirochetes is studied.

Amino Acid Sequence↗
PubMed1972

Treponemal infection in the Australian Northern Territory aborigines.

For many years, a moderate to high prevalence of positive reactions to standard tests for syphilis (STS) has been found in sera from the aboriginal inhabitants of the Northern Territory of Australia. In the survey reported here, people living in 18 areas of the Northern Territory were sampled to determine if the positive STS results were due to treponemal infection and, if so, to what extent this was endemic syphilis, yaws, or venereal syphilis. The results of the Treponema pallidum immobilization test were taken to indicate the presence or absence of treponemal disease in the subject. Clinically, no cases of active treponemal infection were seen, though findings on old infections were difficult to interpret in the older age groups. The prevalence of treponemal infection varied from 3.4% to 58.0% in the areas tested. No serological evidence of treponemal infection in children under 15 years of age was found in 6 areas, while in 12 areas it varied from 2.2% to 32.6%. Sera from 60 people who had no clinical signs of treponemal infection gave high-titre CWR and VDRL test results. It was concluded that yaws, endemic syphilis, and probably venereal syphilis are to be found in the aboriginal population.

Adolescent↗
PubMed1997

Serological markers for treponemal infection in children in rural Kilimanjaro, Tanzania: evidence of syphilis or non-venereal treponematoses?

OBJECTIVE: To determine the seroprevalence of treponemal infection and possible risk factors among children aged 0-14 in the general population of a rural Tanzanian village. METHODS: The survey was conducted as a part of a cross section study of a total village population on HIV and sexually transmitted disease. Among 1708 registered children aged 0-14, the 553 first attending were tested for treponemal infection with both rapid plasma reagin test (RPR) and Treponema pallidum Haemagglutination test (TPHA). These children belonged to a household cohort--also including their parents, siblings, and other household members--with 1339 members; 1224 (91.4% participated in the survey and 82.1% of these were tested for treponemal infection. RESULTS: The overall prevalence for the TPHA test was 6.4% among girls and 1.1% among boys (odds ratio, OR = 6.5; 95% confidence interval, CI: 1.9-22.3). The sex difference was most pronounced in the age group 10-14; 11.1% among girls versus 1.0% among boys (OR = 12.8; CI: 1.6-101.9). Among the 20 children who were TPHA positive, we found two cases of active, congenital syphilis. There was a lack of association between positive serology in children and positive serology in their parents. CONCLUSION: The highly significant predominance of girls testing positive for TPHA, and the concomitant lack of association between parents' and children's serostatus might point to sexual transmission as being the most common route of transmission of treponemal infection in girls during childhood in this village. The sources of infection for the seropositive girls are possibly found outside the family.

Adolescent↗
PubMed2003

Identification of spirochetes (treponemes) in endodontic infections.

The purpose of this study was to determine the prevalence of spirochetes in asymptomatic infected root canals and in endodontic abscesses/cellulitis. Aseptic clinical samples were collected using paper points from 54 infected root canals and from aspirates of 84 abscesses/cellulitis. Oligonucleotide primers were produced for PCR identification of Treponema vincentii, T. pectinovorum, T. medium, T. amylovorum, T. denticola, T. maltophilum, and T. socranskii. PCR detected spirochetes in 51 of 84 (60.7%) samples from abscesses/cellulitis and in 20 of 54 (37.0%) samples from asymptomatic infected root canals. T. socranskii was the most frequently detected (44.9%), followed by T. maltophilum (29.7%), T. denticola (28.9%), T. pectinovorum (13.7%), and T. vincentii (5.1%). The number of treponema species detected ranged from 1 to 5 species per sample. The mean numbers of species detected were 2.3 in abscesses/cellulitis and 2.6 in infected root canals. Significant association among species was found between T. maltophilum and T. socranskii, as well as between T. maltophilum and T. denticola by determining the odds ratio (> 2.0).

Bacterial Typing Techniques↗
PubMed1983

Immune serum confers protection against syphilitic infection on hamsters.

Pooled serum from hamsters immune to syphilitic infection conferred complete protection on recipient hamsters challenged with Treponema pallidum subsp. endemicum. Cutaneous lesions did not develop, and the recipients' lymph nodes weighed less than those of controls and contained no treponemes. Treponemicidal activity in the pooled immune serum was relatively high. When treponemes were incubated in immune serum and complement and the suspension was then inoculated into hamsters, recipients developed neither lesions nor enlarged lymph nodes teeming with treponemes. With hamsters already infected for several weeks, however, immune serum failed to impair or influence the progression of syphilis. Treponemes were eliminated only when immune serum was administered within a short time of syphilitic infection. These results demonstrate that hamsters develop an effective serum-mediated treponemicidal response, but this response is not sufficient to eliminate treponemes at the primary foci of infection.

Animals↗
PubMedDate not supplied

Surface plasmon resonance biosensors for detection of pathogenic microorganisms: strategies to secure food and environmental safety.

This review describes the exploitation of exclusively optical surface plasmon resonance (SPR) biosensors for the direct and indirect detection of pathogenic microorganisms in food chains and the environment. Direct detection is, in most cases, facilitated by the use of defined monoclonal or polyclonal antibodies raised against (a part of) the target pathogenic microorganisms. The antibodies were immobilized to a solid phase of the sensor to capture the microbe from the sample. Alternatively, antibodies were used in an inhibition-like assay involving incubation with the target organism prior to analysis of nonbound antibodies. The free immunoglobins were screened on a sensor surface coated with either purified antigens or with Fc or Fab binding antibodies. Discussed examples of these approaches are the determination of Escherichia coli O1 57:H7, Salmonella spp., and Listeria monocytogenes. Another direct detection strategy involved SPR analysis of polymerase chain reaction products of Shiga toxin-2 genes reporting the presence of E. coli O157:H7 in human stool. Metabolic products have been exploited as biomarkers for the presence of a microbial agent, such as enterotoxin B and a virulence factor for the occurrence of Staphylococcus aureus and Streptococcus suis, respectively. Indirect detection, on the other hand, is performed by analysis of a humoral immune response of the infected animal or human. By immobilization of specific antigenic structures, infections with Herpes simplex and human immunodeficiency viruses, Salmonella and Treponema pallidum bacteria, and Schistosoma spp. parasites were revealed using human, avian, and porcine sera and avian eggs. Bound antibodies were easily isotyped using an SPR biosensor to reveal the infection history of the individual. Discussed studies show the recent recognition of the suitability of this type of instrument for (rapid) detection of health-threatening microbes to food and environmental microbial safety.

Biosensing Techniques↗