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Use of a whole chromosomal probe for identification of Treponema hyodysenteriae.

A whole chromosomal DNA probe labelled with photobiotin was used in a dot blot hybridisation to identify DNA from isolates of Treponema hyodysenteriae, the aetiological agent of swine dysentery. The probe was evaluated using DNA from 13 isolates of T hyodysenteriae and 13 isolates of non-T hyodysenteriae spirochaetes recovered from pigs. The initial test had both a sensitivity and specificity of 92.3 per cent, although when it was repeated the specificity fell to 84.6 per cent. The test was helpful in distinguishing between T hyodysenteriae and other morphologically similar treponemes that are part of the normal flora in the large intestine of pigs. The probe could also be used to detect as little as 10 ng of purified DNA from T hyodysenteriae, or DNA from 2 x 10(6) bacterial cells lysed directly onto nitrocellulose.

Animals↗

HIV, HBV, delta-agent and Treponema pallidum infections in two rural African areas.

In order to compare the seroepidemiology of human immunodeficiency virus (HIV), hepatitis B virus, delta agent and Treponema pallidum infections in two rural populations living in north Uganda (Kitgum district) and in central Burundi (Butezi, Ruyigi region), 448 sera were tested for HBS-Ag, HBS-Ab, and anti-HIV antibodies and screened for syphilis using the T. pallidum haemagglutination (TPHA) test. HBS-Ag positive sera were also tested for anti-delta antibodies. Overall seropositivity rates in healthy subjects, outpatients and inpatients (non-AIDS) were 14.2% and 9.5% in Kitgum district and Butezi, respectively. The prevalence of HBS-Ag and HBS-Ab ranged from 10.0% to 15.6% and from 66.2% to 68.9%, respectively. In north Uganda the rates of anti-delta positivity were 3.1% in the overall population and 30.6% in the HBS-Ag positive subjects. No serum obtained in Butezi was anti-delta positive. In Ugandan people, 64.0% of anti-HIV positive and 25.8% of anti-HIV negative patients were also TPHA-positive (P less than 0.01). For Butezi the corresponding figures were 21.4% and 1.6% respectively (P less than 0.04). On the contrary, no correlation was found between either anti-HIV or TPHA positives and seropositivity for B and delta hepatitis serological markers. The study demonstrated an association between seropositivities for HIV and T. pallidum (TPHA), suggesting common patterns of transmission. On the contrary, no association seemed to exist between HBV and HIV infections.

Adult↗

Pathology of the umbilical cord in congenital syphilis: analysis of 25 specimens using histochemistry and immunofluorescent antibody to Treponema pallidum.

Identification of Treponema pallidum in the placenta is important for diagnosis of congenital syphilis; however, spirochetes are difficult to observe in chorionic villi. To determine the sensitivity of umbilical cord examination for T pallidum, and the association of spirochetes with cord pathology, placentas were prospectively obtained from 25 women with untreated syphilis. The most common finding using hematoxylin-eosin staining was a normal-appearing umbilical cord (48%); necrotizing funisitis was the most frequent pathological lesion (36%). Spirochetes were detected using silver and immunofluorescent staining in 89% of cords, including 92% of histologically normal and 84% of abnormal cords. Three specimens showed subamnionic aggregates of spirochetes, consistent with amniotic fluid infection. Necrotizing funisitis was strongly associated with umbilical artery infection by spirochetes (P = .008). There was a 100% correlation between results of silver and immunofluorescent staining. The umbilical cord is a sensitive site for morphological confirmation of T pallidum; it is significant for the pathologist that spirochetes may often be detected in the absence of overt tissue inflammation or necrosis.

Antibodies, Bacterial↗

Cultivation requirements for Treponema pallidum, Mycobacterium leprae and other microbial and mammalian microaerophilic cells.

Atmospheric and biological evolution progressed simultaneously and today certain cell types flourish only at oxygen tensions which were ambient 600 million years ago, i.e., at 5 to 10 mm Hg. In man, a continuous oxygen flow at these pressures is supplied in the skin where Treponema pallidum, Mycobacterium leprae and members of the genus Rickettsia grow best. In vitro studies support the microaerophilic status of these organisms and of certain other microbial and mammalian cells. Vigorous growth in pure culture will await the development of techniques which can maintain these low oxygen tensions at the cell walls of the microbes as they replicate and consume increasing amounts of oxygen. Continuing failure to consistently isolate microbes from active lesions in patients with rheumatoid arthritis or systemic lupus erythematosus may reflect the universal absence of suitable methods for isolation of microaerophilic microbes.

Anaerobiosis↗

Isolation and characterization of a plasmid from Treponema denticola.

Agarose gel electrophoresis of whole genomic DNA of the oral spirochaete Treponema denticola has revealed a plasmid-like fraction. Purification and restriction enzyme analysis has confirmed the presence of a 2.6-kb circular plasmid, which has been mapped for restriction sites and cloned into the Escherichia coli plasmid pUC18. Southern blot analysis of genomic T. denticola DNA, using the plasmid as a probe, has shown that the plasmid is present only as an extra-chromosomal element. No plasmid-coded recombinant gene product from a PstI insert in pUC18 has been detected in host cells of E. coli by SDS-PAGE or immunoblotting with polyclonal immune rabbit serum to T. denticola. The discovery of this plasmid may provide a useful tool in the application of new molecular approaches in spirochaetal biology.

Blotting, Southern↗

Identification of four new prokaryotic bacterioferritins, from Helicobacter pylori, Anabaena variabilis, Bacillus subtilis and Treponema pallidum, by analysis of gene sequences.

The nucleotide (nt) sequence of the Helicobacter pylori (Hp) napA gene, encoding neutrophil-activating protein A (HPNAP) was determined. Alignment of this sequence with those of known bacterioferritins (Bfr) revealed sequence homology and conservation of a 7-amino-acid (aa) motif constituting the ferroxidase (Frx) center of Bfr in the HPNAP. The N-terminal aa sequence deduced from the iron-regulated mrgC gene of Bacillus subtilis [Chen et al., J. Bacteriol. 175 (1993) 5428-5437] is highly similar to that of HPNAP and contains five Frx center aa residues. The deduced aa sequences for proteins of unknown function in Treponema pallidum [Walfield et al., Infect. Immun. 57 (1989) 633-635] and in the cyanobacterium Anabaena variabilis [Sato, GenBank accession No. JU0384 (1991)] identify these two proteins as Bfr. Although the DNA-binding protein from starved cells of Escherichia coli [Almiron et al., Genes Dev. 6 (1992) 2646-2654] is clearly a HPNAP/Bfr homologue, a significant part of its Frx center is missing. It is unlikely that the intracellular function of HPNAP is related to its ability to activate neutrophils.

Amino Acid Sequence↗

Identification of a fliG homologue in Treponema denticola.

Using a bacteriophage lambda library of Treponema denticola (Td) ATCC 35405 DNA, and, as a reagent, sera derived from individuals with advanced adult periodontal disease, a variety of recombinant clones producing antigens of this oral spirochete have been isolated. Nucleotide sequence analysis of a clone expressing three immunoreactive antigens has revealed the presence of an open reading frame highly homologous to the flagellar switch/motor protein, FliG, which is known to be essential for flagellar assembly and rotation, and chemotaxis in enteric bacteria. The deduced amino-acid sequence of the treponemal FliG protein had 73% similarity (55% identity) to the Bacillus subtilis FliG protein, and showed significant, but lesser homologies to Gram- FliG proteins. Sequence analysis of regions flanking fliG indicated that this gene is immediately preceded by a fliF homologue, further supporting that the cloned DNA encodes FliG of Td. The findings imply that although the signals for control of chemotaxis may be distinctly different in spirochetes, at least some of the molecules involved in torque generation, control of flagellar rotation and signal transduction are highly conserved with other bacteria. The stronger homology of the spirochete FliG with those of Gram+ bacteria is also consistent with recent analyses of other spirochetal genes.

Adult↗

Identification and sequences of the Treponema pallidum fliM', fliY, fliP, fliQ, fliR and flhB' genes.

Information regarding the biology and virulence attributes of Treponema pallidum (Tp) is limited due to the lack of genetic exchange mechanisms and the inability to continuously cultivate this spirochete. We have utilized TnphoA mutagenesis of a Tp genomic DNA library in Escherichia coli (Ec) to identify genes encoding exported proteins, a subset of which are likely to be important in treponemal pathogenesis. We report here the identification and nucleotide (nt) sequence of a 5-kb treponemal DNA insert that contains seven open reading frames (ORFs). The proteins encoded by six of these ORFs have homology with members of a newly described protein family involved in the biogenesis/assembly of flagella and the control of flagellar rotation in Ec, Salmonella typhimurium (St) and Bacillus subtilis (Bs). Certain members of this family are also involved in the export of virulence factors in Yersinia (Yr) spp., St and Shigella flexneri (Sf). We have named these six ORFs fliM', fliY, fliP, fliQ, fliR and flhB'. The operon containing these ORFs has been designated as the fla operon. We hypothesize that the protein products of these genes are involved in the biogenesis/assembly of flagella and the control of flagellar rotation in Tp.

Amino Acid Sequence↗

A mgl-like operon in Treponema pallidum, the syphilis spirochete.

A 38-kDa lipoprotein of Treponema pallidum subsp. pallidum (T. pallidum), the syphilis spirochete, previously was identified as a putative homolog of E. coli MglB [Becker et al. (1994) Infect. Immun. 62, 1381-1391]. In the present study, genome walking in regions adjacent to the T. pallidum 38-kDa lipoprotein gene has identified three contiguous genes (tp-mglB [formerly tpp38], tp-mglA, and tp-mglC) which appear to comprise a mgl-like operon in T. pallidum. A prominent transcript corresponding to tp-mglB, the first gene of the operon which encodes the carbohydrate receptor, is synthesized by T. pallidum along with lesser abundant transcript(s) corresponding to the entire T. pallidum mgl operon. An active promoter 135 bp upstream of tp-mglB is believed to direct mRNA synthesis for the operon. This is the first membrane protein-encoding operon of T. pallidum for which a putative function (glucose import) has been assigned. Furthermore, by analogy with E. coli MglB which interacts with the sensory transducer Trg to induce a chemotactic response, it is possible that T. pallidum also contains a homolog of E. coli Trg or other methyl-accepting chemotaxis proteins. The existence of a mgl operon in T. pallidum thus may have important implications with respect to T. pallidum survival, tissue dissemination, and sensory transduction during virulence expression.

Amino Acid Sequence↗

Treponema hyodysenteriae growth under various culture conditions.

The influence of various culture conditions on the growth of Treponema hyodysenteriae was determined. Six different anaerobically prepared culture broths were tested for the ability to support growth of strains B78, B204 and B169. Each medium contained glucose (0.2%) and 10% (v/v, final concn.) heat-inactivated fetal calf serum. Brain-heart infusion (BHIS), heart infusion (HS) and veal infusion (VS) broths gave the highest cell yields of the spirochete with the shortest incubation times. Vigorous mixing of the cultures and the introduction of O2 (1%, final concn.) into the culture atmosphere were necessary for optimum growth. Although BHIS broth was found to be the best for routine cultivation of the 3 strains, HS broth was more suitable for investigating the physiology of growing cells, inasmuch as cell growth in this medium was limited unless a growth substrate was added. Glucose, fructose, sucrose, galactose, trehalose, N-acetyl-glucosamine, glucosamine, mannose, maltose and pyruvate were growth substrates for all 3 strains. During the growth of B204 cells in HS broth under N2:O2 (99:1), glucose and O2 were consumed and CO2, H2, acetate and butyrate were produced. In HS agar-containing medium, cells of strains B78 and B204 formed spreading colonies typical in appearance to those of other spirochetes.

Animals↗

Typing of Treponema hyodysenteriae by restriction endonuclease analysis.

Restriction endonuclease analysis (REA) was used to type eight well-characterised strains of Treponema hyodysenteriae originating from the U.K., Canada and the U.S.A., and 16 isolates from cases of swine dysentery in Western Australia (W.A.). Several of the W.A. isolates were also serotyped by the method of Baum and Joens (1979), and the two typing techniques were compared. REA typing was more discriminatory than serotyping, being able to distinguish strains within serotypes. The new technique was neither more difficult nor more time-consuming to perform than serotyping. Within the 16 W.A. isolates, three different REA patterns were identified, with common patterns found on different farms. The eight overseas strains had seven different REA patterns, all of which could be distinguished from the patterns of the W.A. isolates.

Animals↗

Biochemical and immunochemical characterisation of strains of Treponema hyodysenteriae.

The protein composition of 18 clinical isolates of Treponema hyodysenteriae from pigs with swine dysentery in Australia were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. Coomassie Blue stained SDS-PAGE-profiles of whole cell and outer membrane (OM) proteins demonstrated the same gel pattern among the T. hyodysenteriae isolates, particularly the OM proteins in the molecular mass (Mr) range of 30 kDa to 40 kDa. The T. hyodysenteriae isolates were categorised into two distinct groups (A and B) based on the strain-variability in the 37 kDa OM protein. Immunoblotting of whole cell proteins after SDS-PAGE using serum from rabbits and pigs immunised with known T. hyodysenteriae serotypes revealed a number of common immunoreactive bands in all isolates. LPS typing of the T. hyodysenteriae isolates by immunoblotting with the rabbit antiserum revealed one additional serotype emphasising the LPS heterogeneity among the strains isolated from geographic locations in Australia, Great Britain and the U.S.A. Immunoblotting of the OM preparations revealed several common immunoreactive polypeptides corresponding to Mr values of 34 kDa to 30 kDa among the T. hyodysenteriae and T. innocens isolates but a distinct 39 kDa found only in the T. hyodysenteriae isolates. Trypsin proteolysis of intact. T. hyodysenteriae cells caused selective loss of these and other major abundant proteins identifying the location of the 39 kDa, 36 kDa, 34 kDa and 30 kDa proteins on the cell surface and suggesting a possible role of these proteins in the pathogenesis of swine dysentery.

Animals↗

Multilocus enzyme electrophoresis for identification and typing of Treponema hyodysenteriae and related spirochaetes.

An assessment was made of multilocus enzyme electrophoresis as a means of identifying and typing spirochaetes isolated from pigs. Using five enzyme systems, 36 isolates from Australia, the U.K. and the U.S.A. were divided into 12 electrophoretic types or multilocus genotypes, comprising four major, genetically distinct groups. All 26 isolates of Treponema hyodysenteriae fell into one group, members of which showed relatively little genetic diversity. Ten isolates of non-pathogenic spirochaetes fell into three genetically different groups. Although the technique was capable of typing organisms within the groups, it was not always as discriminatory as DNA-restriction endonuclease analysis. Examination of additional enzyme loci should increase the sensitivity of the method for typing and for overall assessment of genetic relationships between spirochaetes.

Alleles↗

Typing of Australian isolates of Treponema hyodysenteriae by serology and by DNA restriction endonuclease analysis.

A total of 91 isolates of Treponema hyodysenteriae which were obtained from 62 piggeries located around Australia were typed by serology and by DNA restriction endonuclease analysis (REA). The isolates fell into eight serogroups, of which groups B and D were the most common. Isolates with different REA patterns were recognised within serogroups, whilst a few isolates with the same REA pattern were placed into different serogroups. Some of the latter isolates were either from the same piggery or from farms with epidemiological links, thus indicating the bacteria may have altered their antigenic properties. A total of 31 different REA patterns were recognised amongst the Australian isolates. These comprised eight major patterns, with four of these being subdivided on the basis of minor differences in banding. Where a number of isolates were obtained from individual piggeries these all had the same REA pattern, and in one piggery isolates with the same pattern were recovered over a five year period. Plasmid bands were observed in 70 of the Australian isolates (77%), and in six of the seven overseas isolates included in the study for comparison. These plasmids did not affect the REA pattern. Of the States from which substantial numbers of isolates were examined, the greatest number of different strains (12 amongst 19 piggeries) were found from Victoria, and there were 10 REA patterns in strains from 24 piggeries in Queensland. Despite the large total number of different strains of T. hyodysenteriae in Australia, only three were found in more than one State.

Animals↗

Purification of Treponema pallidum, Nichols strain, by two-step column chromatography.

A rapid and simple purification method for Treponema pallidum, Nichols strain, the etiological agent of venereal syphilis, was developed. A 40-ml suspension of organisms (1.10(9)/ml) was extracted from rabbit testicular tissue and solubilized with a non-ionic detergent, 1-O-n-octyl-beta-D-glucopyranoside. Solubilized antigens were purified by cation-exchange and hydroxyapatite column chromatography. The overall recovery of immunoreactive material was 48.3% and the specific activity increased. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting analysis confirmed the purity and species specificity of the purified antigen.

Animals↗

Characterization of lipid-modified immunogenic proteins of Treponema pallidum expressed in Escherichia coli.

This study describes the sequence of the immunodominant Treponema pallidum surface protein TpD and its expression in Escherichia coli. The translated TpD DNA sequence revealed the presence of a putative site for lipid-modification downstream from the signal sequence of this membrane protein. Growth of TpD-expressing E. coli in the presence of radioactive palmitic acid revealed that TpD was lipid-modified. Three other, previously characterized cloned proteins of T. pallidum were also lipid-modified. The N-termini of two of three sequenced T. pallidum proteins contain a tetrapeptide sequence characteristic for lipoproteins in Gram-negative bacteria: Leu-X-Y-Cys. Only TpD differed from this consensus sequence in the substitution of the first residue by Phe. The apparent high incidence of lipoproteins among E. coli recombinants expressing T. pallidum antigens suggest an important role of lipoproteins in the induction of humoral immunity during syphilitic infection.

Amino Acid Sequence↗

Production and characterization of monoclonal antibodies to Treponema pallidum.

This study was attempted to produce the monoclonal antibodies specific for Treponema pallidum and to investigate their characteristics, thereby contributing to identify the antigenic structure and to apply the diagnosis of syphilis. The seven clones (YS 3, YS 75, YS 307, YS 481, YS 343, YS 1 and YS 406) secreting monoclonal antibodies reactive with T. pallidum were produced. The isotypes of the seven monoclonal antibodies produced were defined. Optical densities of the 7 monoclonal antibodies were ranged from 0.59 to 1.48 as measured by ELISA. Monoclonal antibodies from 5 of the 7 clones which could agglutinate sheep RBC sensitized with T. pallidum, showed strong fluorescences. The monoclonal antibody from YS 343 was cross-reactive with non-pathogenic treponemes, but the other 6 monoclonal antibodies reacted with T. pallidum specifically. On immunoblotting, monoclonal antibodies from 5 of the 7 clones reacted with a polypeptide of a molecular weight of 47 kDa, and monoclonal antibodies from YS 343 reacted with a polypeptide of 64 kDa common to both T. pallidum and T. phagedenis. The above results revealed that 7 clones secreting monoclonal antibodies reactive to T. pallidum were produced successfully, and specific antibodies reacting with major antigenic polypeptides of a molecular weight of 47 kDa would be useful in the diagnosis of syphilis in the future.

Animals↗

Proteolytic and oxidoreductase activity of Treponema denticola ATCC 35405 grown in an aerobic and anaerobic gaseous environment.

The cells of a human oral spirochete, Treponema denticola ATCC 35405, and of seven clinical isolates of this organism obtained from the subgingival dental plaque of periodontitis patients were studied for their ability to grow in an aerobic and an anaerobic environment, and for their profile of peptidohydrolase and oxidoreductase enzymes. The growth yield of aerobically grown cultures was either comparable to or higher than that of anaerobically grown ones regardless of whether prereduced broth, freshly prepared broth or oxidized broth was used. However, elimination of certain supplements from the growth media resulted in poor growth regardless of the nature of the gaseous environment. The microscopic morphology and motility of the cells were not affected by differences in the gaseous atmosphere. Quantitative studies on several peptidohydrolase activities suggest that anaerobically grown cells displayed higher specific activity especially toward N alpha-L-prolyl-2-naphthylamine, indicating that increased synthesis of proline iminopeptidase enzymes (or enzyme) of the cells was associated with anaerobic growth conditions. The formation of enzymes hydrolysing N alpha-benzoyl-DL-arginyl-2-naphthylamine (and the corresponding p-nitroaniline) was not affected to the same extent. Growth experiments suggest that T. denticola ATCC 35405 is a facultatively anaerobic spirochete instead of an obligate anaerobe as reported in previous literature. The quantitative enzyme studies suggest that the gaseous growth atmosphere of the cells can exert a selective effect on the activity levels of certain peptidolytic enzymes of this organism. Such effects were not observed when the whole cells were studied by means of qualitative or semi-quantitative enzyme tests. The activities of catalase, peroxidase and superoxide dismutase of the cells were low and variable. Because of this, it was not possible to relate these oxidoreductase activities to the composition of the gaseous atmosphere.

Aerobiosis↗