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PubMed2007

Colonic amoebiasis and spirochetosis: morphological, ultrastructural and microbiological evaluation.

BACKGROUND: The present study reports on a prompt diagnosis of colonic amoebiasis with colonic spirochetosis by Brachyspira aalborgi and B. pilosicoli; such diagnosis allowed exclusion of other diseases and resolution of the case after specific treatment. METHODS AND RESULTS: A 37-year-old Italian man with a history of several months' mucosal diarrhea travelled to Greece, Romania and Tunisia. After his last trip he presented with an increase of up to 3-5 discharges daily, associated with bloody diarrhea, supporting the clinical suspect of inflammatory bowel disease. Colonoscopy revealed erosions from the cecum to the rectum, and ulcers both in the descending and sigmoid colon. Structures resembling amoebic trophozoites and sinusoidal microorganisms were observed in the colonic biopsies at histopathology and electron microscopy. Entamoeba histolytica DNA was detected by small-subunit rDNA polymerase chain reaction (PCR) from feces, rectal biopsies and isolated trophozoites. Spirochetes were identified from feces, colonic biopsies and cultures using a 16S rDNA restriction fragment length polymorphism-PCR specific for the detection of B. aalborgi and B. pilosicoli. After therapy, the patient was restored to health. CONCLUSIONS: The rapid identification of E. histolytica, B. aalborgi and B. pilosicoli using traditional and specific and sensitive molecular methods permitted an accurate diagnosis and a specific therapy. It is suggested that mixed infection by parasites and spirochetes might occur more frequently than expected: it would be of extreme interest and importance to intensify clinical findings, and one infection should not prompt the pathologist/clinician to stop looking.

Adult↗
PubMed2000

Presence of 22-kDa protein reacting with sera in piglets experimentally infected with Brachyspira hyodysenteriae.

In order to examine the effect of spectinomycin on outbreaks of swine dysentery, experimental infection of piglets with Brachyspira hyodysenteriae was carried out. Feed with and without spectinomycin (SP) was given to each piglet ad libitum and the susceptibility of the piglets to infection with B. hyodysenteriae was compared between SP-treated and untreated piglets. The results showed that the SP-treated piglets did not display clinical signs of swine dysentery unlike the untreated piglets. The sera obtained from these piglets were examined by the microscopic agglutination test and antibodies to B. hyodysenteriae in both groups of experimentally infected piglets were detected and the reaction was serogroup-specific. The agglutination titers were very high in the untreated piglets with dysentery while the titers in the SP-treated piglets were lower than those in the untreated piglets. In addition, the immunoblotting technique was applied and the results demonstrated that 22- and 17-kDa proteins in strain ATCC 31212 (serogroup B) reacted strongly with the sera from the untreated piglets but not with the sera from the SP-treated piglets. The 22- and 17-kDa proteins also reacted with strain ATCC 27164 (serogroup A) which belongs to a different serogroup. The 22- and 17-kDa proteins were also confirmed in six other strains of B. hyodysenteriae which belong to six different serogroups. These proteins were sensitive to proteinase K. These results indicate that the 22- and 17-kDa proteins are common to eight strains of B. hyodysenteriae which differ serologically from each other.

Agglutination Tests↗
PubMed1994

16S rDNA sequence and phylogenetic position of an uncultivated spirochete from the hindgut of the termite Mastotermes darwiniensis Froggatt.

We have analyzed the 16S rDNA sequence and the phylogenetic position of an uncultivated spirochete from the hindgut contents of the Australian termite Mastotermes darwiniensis Froggatt. The 16S rRNA genes of bacteria from the hindgut contents of Mastotermes darwiniensis were amplified by polymerase chain reaction. The amplification products were cloned and sequenced. The sequences were compared to known homologous primary structures. Two of the clones (MDS1 and MDS3) had an insert of 1498 nucleotides showing typical signatures of spirochete 16S rRNA sequences. The sequences of the two clones were most similar to the 16S rRNA sequence of Spirochaeta stenostrepta (89.8%) and Treponema sp. strain H1 (90.7%). Phylogenetical analysis positioned the hindgut spirochete sequence with that of the free-living anaerobic Spirochaeta stenostrepta and Treponema sp. strain H1 as its nearest relatives within the cluster of the spirochetes. We conclude that analyzed SSU rDNA sequences originate from a spirochete related to the genus Treponema. It is possibly one of the uncultivated unique spirochetes symbiotic in termite hindguts.

Animals↗
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↗
PubMed1995

A monoclonal antibody reacting with the cell envelope of spirochaetes isolated from cases of intestinal spirochaetosis in pigs and humans.

A monoclonal antibody (mAb) designed BJL/AC1 was prepared against the cell envelope of an intestinal spirochaete (strain 3295) that was isolated from a pig with intestinal spirochaetosis. The mAb reacted with a band of approximately 29 kDa in cell envelope preparations from 13 porcine and 11 human spirochaetes isolated from cases of intestinal spirochaetosis, but did not react with preparations made from a range of other intestinal spirochaetes. Immunogold labelling demonstrated that the reactive epitope was located on the cell envelope of the strains causing intestinal spirochaetosis. The mAb was used in an indirect immunofluorescence test to detect spirochaetes in the faeces of pigs with experimentally induced intestinal spirochaetosis. The mAb should prove to be a useful reagent for detection and identification of spirochaetes that are specifically associated with intestinal spirochaetosis.

Animals↗
PubMed1996

Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons.

Multilocus enzyme electrophoresis (MEE) analysis and comparisons of nearly complete 16S rRNA gene sequences (1416 nucleotide positions) were used to evaluate phylogenetic relationships among Serpulina hyodysenteriae strain B78T, S. innocens strain B256T, Brachyspira aalborgi strain 513AT, and eight uncharacterised strains of swine, avian, and human intestinal spirochaetes. From MEE analysis, nine strains could be assigned to five groups containing other intestinal spirochaetes (genetic distances between groups = 0.6-0.9). Chicken spirochaete strain C1 and B. aalborgi 513AT represented unique electrophoretic types and formed their own MEE groups. Despite MEE differences, the 11 strains had highly similar (96.3-99.9%) 16S rRNA sequences. These findings point out limitations of both MEE analysis and 16S rRNA sequence comparisons when used as solitary techniques for classifying intestinal spirochaetes related to Brachyspira/Serpulina species.

Alleles↗
PubMed1996

Phenotypic characteristics of Serpulina pilosicoli the agent of intestinal spirochaetosis.

The phenotypic characteristics of three Serpulina pilosicoli strains isolated from humans with diarrhoea (WesB, Kar, Hrm7) and two porcine S. pilosicoli strains isolated from pigs with intestinal spirochaetosis (1648, 3295), were compared with the type strain of the species P43/6/78T (T = type strain) and other intestinal spirochaetes within the genus Serpulina. All S. pilosicoli strains had a characteristic ultrastructural appearance, displayed similar growth rates, hydrolysed hippurate, lacked beta-glucosidase activity, utilised D-ribose as a growth substrate, and had similar sensitivities to rifampicin and spiramycin. The only consistent phenotypic characteristic that differentiated human strains from porcine strains of S. pilosicoli was that the human strains all utilised the pentose sugar D-xylose. These distinguishing phenotypic traits appear useful for identifying S. pilosicoli.

Adult↗
PubMed1997

Spirochaetes and other bacterial species associated with bovine digital dermatitis.

The 16S rRNA genes from spirochaetes associated with digital dermatitis of British cattle were amplified by polymerase chain reaction from digital dermatitis lesion biopsies using one universal and one treponeme-specific primer. Two treponemal sequences were identified both of which shared a high degree of homology with the oral pathogen Treponema denticola (98%). Two further 16S rRNA gene sequences were obtained and shared similarity to Bacteroides levii (99%) and Mycoplasma hyopharyngis (98%). Polymerase chain reaction with T. denticola-specific primers amplified a potential virulence gene from digital dermatitis lesions which shared a high degree of homology to the 46-kDa haemolysin gene of T. denticola. The significance of the presence of organisms in digital dermatitis lesions of the bovine foot which are closely related to oral pathogens is discussed.

Amino Acid Sequence↗
PubMed2002

Identification of Brachyspira hyodysenteriae-specific DNA fragments using representational difference analysis.

Two novel Brachyspira hyodysenteriae-specific DNA fragments, designated as Bh100 and Bh400, were identified using representational difference analysis. To isolate the fragments the combined DNA of the Brachyspira pilosicoli, Brachyspira intermedia, Brachyspira murdochii and Brachyspira innocens reference strains was subtracted from the genome of B. hyodysenteriae strain B204. Both fragments were present in a single copy and mapped to different positions on the genome of B. hyodysenteriae B78(T). Larger fragments encompassing the continuous open reading frames (ORF) of Bh100 and Bh400 were cloned and analysed. Whereas the ORF of 2130 bp encompassing Bh100 did not show homology to any known bacterial protein, Bh400 was part of a putative operon with significant homology to the phosphotransferase system of Bacillus subtilis.

Blotting, Southern↗
PubMed2006

Spherical body formation in the spirochaete Brachyspira hyodysenteriae.

When cultures of Brachyspira hyodysenteriae were grown under a wide range of in vitro conditions, at least 1% of the cells formed spherical bodies different to the normal helical form. This percentage increased considerably in aging cultures or following their incubation in caramelized media. Spherical body formation was initiated from a terminal localized swelling of the outer sheath followed by a retraction of the protoplasmic cylinder into the resulting swollen vesicle. As this occurred, the periplasmic flagella seemed to unwind from the protoplasmic cylinder. Once retracted, the protoplasmic cylinder was found to be wrapped in an organized manner around the inner surface of the membrane of the swollen vesicle. Although most were 2-3 microm in diameter, some much larger spherical bodies (6-12 microm diameter) were occasionally seen, with a corresponding increase in the visible number of peripheral protoplasmic cylinder cross-sections. Spherical bodies from older cultures did not contain protoplasmic cylinders arranged around the periphery, but instead were characterized by the presence of a centrally located, electron-dense body c. 0.5-0.8 mum in diameter. Brachyspira hyodysenteriae spherical bodies differ in both their structural organization and probable method of formation from similar structures described in other spirochaete genera.

Animals↗
PubMed1979

Periodontal therapy by local delivery of tetracycline.

The present investigation assessed the feasibility of treating periodontal disease by controlled delivery of antibacterial agents from within periodontal pockets. Tetracycline-filled hollow fibers placed in the gingival sulcus were shown to have a dramatic effect both on the periodontal microflora and clinical manifestations of disease. Furthermore, it was found that drug-filled cellulose acetate hollow fibers are biologically compatible with the environment and can be manipulated by dental personnel to provide drug therapy with less than 1/1000 the amount of tetracycline that would have been used for systemic therapy. Of theoretical importance is the observation that virtual elimination of spirochetes from the gingival sulcus is possible by a single placement of tetracycline-filled hollow fibers, and spirochetes, once eliminated from a site, do not rapidly recolonize despite the persistence of viable organisms elsewhere in the mouth.

Delayed-Action Preparations↗
PubMed1981

Treatment of periodontal infections due to anaerobic bacteria with short-term treatment with metronidazole.

In the present report, five selected periodontal patients were treated for 1 week with metronidazole. Two of the patients had their teeth scales and root-planed the week they received metronidazole. Prior to treatment, B. asaccharolyticus accounted for 41% of the cultivable isolates and the spirochetes averaged 29% of the microscopic count in plaque removed from each of four pockets per patient. The presence of these elevated proportions of periodontopathic bacteria combined with the presence of periodontal pockets and attachment loss suggested that the patients were in a state of an active infectious process involving primarily anaerobic bacteria. If this be the case, then antimicrobial therapy directed against these anaerobes wih metronidazole was indicated. The 1-week treatment with metronidazole significantly reduced the proportions of these organisms for up to 6 months after treatment. Coincident with these findings was an improvement in the clinical parameters, especially in those sites that initially had greater than 5 mm pocket or attachment loss. These sites showed a 2 mm or more reduction in pocket depth and an almost 2 mm gain in apparent attachment that was evident 6 months after treatment. The results obtained were in only five patients. However, the magnitude of improvement suggests that antimicrobial therapy directed against anaerobic organisms may be a valuable adjunct to periodontal therapy.

Adolescent↗
PubMed1981

The predominant cultivable dental plaque flora of beagle dogs with periodontitis.

The predominant cultivable dental plaque flora was studied in 10 adult female beagle dogs with advanced periodontitis. Supragingival and subgingival plaque from a maxillary third premolar (P3) was removed and cultured anaerobically on various growth media and all colonies were subcultured and partially characterized. Histopathological specimens of the plaque sampling sites showed significant loss of connective tissue attachment. Spirochetes were found in all samples. Anaerobic gram-negative organisms were predominant in both types of plaque accounting for about 55% of the cultivable organisms in the supragingival plaque and almost 75% in the subgingival plaque. Bacteroides asaccharolyticus was the most predominant organism in the supragingival plaque, whereas Fusobacterium nucleatum predominated in the subgingival flora. Streptococcal and actinomycotic species were common in the supragingival plaque, but their proportions, especially those of the actinomycetes, were decreased in the subgingival flora. In many respects the bacterial profile associated with disease resembled that reported in human periodontal disease.

Actinomyces↗
PubMed1981

Clinical and antimicrobial effect of stannous fluoride on periodontitis.

The antimicrobial effect of stannous fluoride (SnF2) on suspected pathogenic morphotypes of bacterial plaque in chronic periodontal disease was assessed. Alterations in morphological groups of subgingival and supragingival plaque bacteria from three diseased sites and one periodontally healthy site were determined by darkfield microscopy in 10 male patients with advanced periodontitis. In addition, clinical indices were also measured. The three diseased sites received either 1.64% SnF2, 0.4% SnF2 or sterile saline by subgingival irrigation. The healthy site received sterile saline only. The patients were seen weekly over a 10-week period and at each appointment bacterial sampling and clinical indices were measured. Results indicated that 1.64% SnF2 caused a dramatic and sustained decrease of subgingival motile bacteria and spirochetes following irrigation. By the sixth week these bacteria returned to 50% of their original concentration. Bleeding index scores, which were significantly reduced, correlated positively to the reduction in motile bacteria and spirochetes. Sites irrigated with 0.4% SnF2 also demonstrated a similar pattern of reduction of motile bacteria and spirochetes but without their total elimination and a more rapid return to original levels. In the diseased sites receiving sterile saline there was a decrease in motile bacteria and spirochetes, corresponding with saline irrigation, which rapidly returned to baseline levels. The healthy control sites were unchanged. Supragingival plaque index scores did not correlate with the bleeding index, motile bacteria and spirochetes, or with subgingival irrigation.

Adult↗
PubMed1985

Sampling of subgingival microbiota for dark-field microscopy.

The aim of the present investigation was to study the validity of 2 different methods of sampling the subgingival microbiota. The composition of the flora was estimated using the dark-field microscope. Subgingival samples were obtained using either a curette or a Hamilton microsyringe for a subgingival washing. The method of determination of the composition of the subgingival microorganisms was found to be very reproducible when the same investigator performed the counting. Samples obtained by the washing technique had a slight but significantly higher proportion of coccoid cells when compared to samples obtained by curette. The calculation of the reproducibility of the washing technique as assessed in 6 immediately repeated samples from the same sites yielded a coefficient of variation of 40% for spirochetes and motile rods. When 9 repeated samples from each of 28 sites (14 with a curette, 14 with washing) were studied over a period of 32 days, no systemic alterations were observed in the % of spirochetes and motile rods. The variation within each individual site was high. For curette samples and washing samples, the coefficients of variation for spirochetes and motile rods were 85% and 63%, respectively. The present investigation clearly demonstrated that care should be taken in the interpretation of single samples of subgingival microbiota. A more relevant picture of the actual situation is achieved when trends of repeated samples over time are studied.

Bacteria↗
PubMed1985

Antimicrobial irrigation of deep pockets to supplement non-surgical periodontal therapy. II. Daily irrigation.

106 sites with probing pocket depths 7 mm or greater from 14 patients were treated with plaque control instruction and 1 episode of root planing. Sites in each patient were either irrigated with 2% chlorhexidine or left as non-irrigated controls. Irrigation immediately followed root planing and was repeated daily, by the patient, for 24 weeks. Clinical measurements were made at 12 and 24 weeks, as were gingival washings for determining the number and % of spirochetes. Results at 24 weeks demonstrated that bleeding scores decreased from 91% to 9%; the % of spirochetes dropped from approximately 9% to less than 1%; probing pocket depths decreased from 7.5 to 4.5 mm, and probing attachment levels gained 1.1 to 1.4 mm. The chlorhexidine irrigated experimental group and the non-irrigated control group did not differ significantly in any of the studied parameters. Thus, daily patient-administered chlorhexidine irrigation of deep pockets did not augment the effects of non-surgical periodontal therapy.

Adult↗
PubMed1985

Subgingival spirochete and leukocyte counts as indicators of response to therapy.

The aim of this study was to determine whether changes in probing attachment levels are related to subgingival spirochete or leukocyte counts in periodontal pockets. Following initial clinical recordings and therapy consisting of oral hygiene instruction and root debridement, the probing attachment levels of proximal sites of 120 single-rooted teeth in 7 patients were measured every 3 months for 12 months. The measurements of each site were subjected to regression analysis, which determined whether the site was deteriorating, improving, or non-changing. Subgingival washings were taken of 19 deteriorating, 22 improving, and 127 non-changing sites to determine the number and % of spirochetes and the number of leukocytes at each site. Improved probing attachment levels were associated with reduced numbers of spirochetes and leukocytes. However, the ranges of individual measurements of subgingival washing variables overlapped considerably between groups. Spirochete and leukocyte counts related better to the 12-month probing depths than to changes in probing attachment levels during the preceding 12 months. These findings suggest that none of the tested subgingival washing parameters are suitable indicators of changes in attachment levels on an individual site basis.

Gingiva↗