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PubMed1997

Recognition of two new species of intestinal spirochetes: Serpulina intermedia sp. nov. and Serpulina murdochii sp. nov.

On the basis of DNA-DNA hybridization data, nine intestinal spirochete strains were grouped into five genospecies. Three of these genospecies were previously recognized Serpulina species, Serpulina hyodysenteriae (type strain, B78), Serpulina innocens (type strain, B256), and Serpulina pilosicoli (type strain, P43/6/78; previously "Anguillina coli"). The other two genospecies were found to be new Serpulina species, for which we propose the names Serpulina intermedia sp. nov. (with type strain PWS/A) and Serpulina murdochii sp. nov. (with type strain 56-150). S. intermedia and S. murdochii cells had a typical spirochete ultrastructure with 22 to 28 periplasmic flagella per cell. Various soluble sugars were growth substrates for S. intermedia and S. murdochii. During growth in basal heart infusion broth supplemented with fetal calf serum beneath an O2-N2 (1:99) atmosphere, cells of these new species consumed oxygen and glucose and produced H2, CO2, acetate, butyrate, and ethanol. The G + C content of the DNA of S. murdochii 56-150T was 27 mol%, and the G + C content of the DNA of S. intermedia PWS/AT was 25 mol%. In addition, a restriction fragment length polymorphism-PCR assay for the detection of intestinal spirochetes was developed. The assay was based on generation and restriction endonuclease analysis (with HinfI, TaqI, Sau3A, and MboII) of a 558-bp amplicon of ribosomal DNA (rDNA) encoding 16S rRNA. The PCR amplification was specific for Serpulina species and Brachyspira aalborgi. Four restriction digest patterns were found for the five Serpulina species. HinfI restriction differentiated S. murdochii and S. innocens from the other species. Sau3A and TaqI restrictions gave unique fragment patterns for S. murdochii and S. pilosicoli, respectively. S. hyodysenteriae and S. intermedia DNAs gave the same fragment pattern regardless of the enzyme tested. B. aalborgi was differentiated from the Serpulina species by MboII digestion of the 16S rDNA amplicon.

Bacterial Proteins↗
PubMed1998

Variation amongst human isolates of Brachyspira (Serpulina) pilosicoli based on biochemical characterization and 16S rRNA gene sequencing.

Brachyspira pilosicoli (formerly Serpulina pilosicoli) causes swine spirochaetosis and can also be isolated fro human faeces, although its role in human disease remains unclear. The genetic and biochemical variations amongst 19 isolates of human spirochaetes from five different countries were evaluated and compared to those found amongst swine isolates of B. pilosicoli. All isolates were negative for beta-glucosidase and all but one were positive for hippurate hydrolysis, which are characteristics typical of B. pilosicoli. The isolates showed variation in indole production and alpha-galactosidase and alpha-glucosidase activity, other characteristics which can be used to identify B. pilosicoli. The DNA sequences of part of the 16S rRNA gene differed from each other and from that of B. pilosicoli by 0-3 bp out of 283 bp. It is concluded that there is considerable variation amongst human intestinal spirochaetes. Since few of the isolates reported here match the current criteria for B. pilosicoli, it is concluded that this species is more heterogeneous than previously appreciated. However, it cannot be excluded that some isolates may belong to uncharacterized related Brachyspira/Serpulina species.

Animals↗
PubMed2000

Evaluation of blood culture systems for detection of the intestinal spirochaete Brachyspira (Serpulina) pilosicoli in human blood.

The anaerobic intestinal spirochaete Brachyspira (Serpulina) pilosicoli has been isolated from the bloodstream of French patients by manual blood culture systems. The purpose of this study was to determine whether the automated and manual blood culture systems used in Australia are suitable for growth and detection of this organism. Strains of B. pilosicoli were added to human blood to give concentrations ranging from 1 x 10(4) to 1 x 10(1) spirochaetes/ml and 10-ml volumes were inoculated into the media. Three strains of B. pilosicoli grew slowly in all manual Hemoline and BBL Septi-Chek formulations tested. Subcultures taken between 2 and 10 days after inoculation yielded growth only after incubation for a further 5-8 days. Growth and automated detection were achieved in the BACTEC system with Anaerobic/F medium with or without Fastidious Organism Supplement. Minimum time to signal for nine strains varied between 5.6 and 14.9 days, with a minimum concentration of 10(1) spirochaetes/ml of blood being detected. None of nine strains gave a positive signal in the BacT/Alert system when FAN Anaerobic culture bottles were used; however, four strains were detected by subculture taken at 7 or 14 days after inoculation. When Anaerobic medium was used in the BacT/Alert system, two of three strains gave a signal and the other strain grew and was detected by subculture. Spirochaetaemias caused by B. pilosicoli may be unrecognised because detection time by the signal or subculture exceeds 5 days.

Bacteremia↗
PubMed1987

beta-Lactamase production by intestinal spirochaetes.

beta-Lactamase production was demonstrated in four of nineteen strains of intestinal spirochaetes isolated from human subjects. The enzyme was preferentially active against penicillins and was inhibited by clavulanic acid; it was membrane bound and non-inducible. No plasmids were detected in the intestinal spirochaetes and the beta-lactamase-production characteristic was not transferable to non-producing strains.

Humans↗
PubMed1992

The periplasmic flagella of Serpulina (Treponema) hyodysenteriae are composed of two sheath proteins and three core proteins.

The major components of the periplasmic flagella of the spirochaete Serpulina (Treponema) hyodysenteriae strain C5 were purified and characterized. We demonstrate that the periplasmic flagella are composed of five major proteins (molecular masses 44, 37, 35, 34 and 32 kDa) and present their location, N-terminal amino acid sequence and immunological relationship. The 44 kDa and the 35 kDa protein are on the sheath of the periplasmic flagellum, whereas the 37, 34 and 32 kDa protein reside in the periplasmic flagellar core. The two sheath flagellar proteins are immunologically related but have different N-terminal amino acid sequences. The N-terminus of the 44 kDa protein shows homology with the sheath flagellins of other spirochaetes, but the 35 kDa protein does not. The three core proteins are immunologically cross-reactive and their N-terminal amino acid sequences are almost, but not completely, identical, indicating that the core proteins are encoded by three distinct genes. The core proteins show extensive N-terminal sequence similarities and an immunological relationship with periplasmic flagellar core proteins of other spirochaetes.

Amino Acid Sequence↗
PubMed1997

Evidence for Serpulina hyodysenteriae being recombinant, with an epidemic population structure.

The population structure of Serpulina hyodysenteriae was investigated using multilocus enzyme electrophoresis. A total of 231 isolates were divided into 50 electrophoretic types (ETs), with a mean genetic diversity of 0.29 for the number of ETs and 0.23 for the number of isolates. Subsets of isolates from two Australian states (71 isolates from Victoria and 68 isolates from Queensland) exhibited as much genetic variation as the entire collection. The calculated index of association (IA) for the number of ETs (0.29 +/- 0.17) was not significantly different from zero, and hence provided evidence for the occurrence of significant genetic recombination accounting for the observed variation between strains. In contrast, the IA for the number of isolates (3.93 +/- 0.03) was significantly different from zero, with seven of the 50 ETs (ETs 4, 6, 13, 14, 20, 33 and 35) containing 51% of all the isolates. Even when multiple isolates from the same farm were removed from the analysis, the IA value for the number of isolates remained significantly greater than zero (IA 9.87 +/- 0.04), indicating that it was not biased by their inclusion. The results suggest that S. hyodysenteriae has an epidemic population structure.

Animals↗
PubMed1985

Observations by light microscopy and transmission electronmicroscopy on intestinal spirochaetosis in baboons (Papio spp.).

Spirochaetes were recognised in the large bowel of 57 of 59 baboons as a basophilic fringe at the microvillous brush border of the epithelial cells. Both caecum and colon were usually affected, but seven animals had spirochaetes in the caecum alone. Examination of three animals by transmission electronmicroscopy revealed only one type of spirochaete; ring forms and cross-walls were present. Inflammatory changes were not seen in association with the infection, and the distribution of spirochaetes in 10 animals with soft or diarrhoeic faeces resembled that in normal animals.

Animals↗
PubMed1987

Demonstration of spirochaetes in patients with Lyme disease with a modified silver stain.

Spirochaetes were demonstrated in material from patients with Lyme disease by short-time high-concentrate silver impregnation after treatment with amylase. Removal of mucoid material was essential to visualise Borrelia burgdorferi. Lyme spirochaetes were demonstrated in material from 23 patients with Lyme disease--erythema chronicum migrans (ECM) 10, lymphadenosis benigna cutis (LABC) 7, arthritis 4 and Bannwarth's syndrome 2. Spirochaetes were localised in the subepidermal zone, peri- and intravascularly, and in collagen fibres in ECM and LABC, and beneath the synovial lining cells in arthritis, producing marked vascular changes with fibrosis and synovial hyperplasia. Spirochaetes were also demonstrated in CSF from a patient with Bannwarth's syndrome.

Animals↗
PubMed1997

The challenge of growing oral spirochaetes.

Research in periodontal disease has shown the presence of oral spirochaetes repeatedly in subgingival plaque. There is uncertainty as to whether these spirochaetes are involved in the actual disease process; however, it has been shown that their presence is a definite marker for disease occurrence. An understanding of their role in periodontal disease requires further characterisation of these organisms. Diagnostic tests would be useful for the clinician and enable treatment for the patient to be planned. Studies on characterising the different treponemal species have been limited by difficulties in culturing these organisms. Moreover, there is a need to obtain pure cultures of these organisms and to identify them in order to associate particular species with disease and, ultimately, to make probes for their easy detection directly from dental plaque. This review examines the methods used, and reports our own experience, in obtaining pure cultures of oral spirochaetes. The techniques available and the problems that occur when identifying these organisms are also considered.

Bacteriological Techniques↗
PubMed1998

Morphologic and temporal characterisation of lesions in an enhanced murine model of Serpulina hyodysenteriae infection.

This laboratory has previously reported a murine model of Serpulina hyodysenteriae infection in which mice fed a defined diet, Teklad 85420 (TD), developed caecal lesions more consistently than mice fed a conventional rodent chow (CRC). The objectives of the current studies were to characterise and compare the time of onset of lesions, the morphological nature and severity of lesions and the extent of colonisation by S. hyodysenteriae in mice fed the two diets. In the first of two experiments, 50 C3H/HeJ and 50 C3H/HeOuJ mice were fed either TD or CRC and then half of each group was infected with S. hyodysenteriae. Mice (n = 5) from each group were killed and examined on days, 1, 2, 4, 9 or 17 after infection. Each mouse was examined grossly and microscopically and assigned lesion scores based on lesion severity. The second experiment was designed in an identical way to the first, but had slightly smaller group sizes (n=20). Mice (n=4) were killed for necropsy at the same five time points after infection and their caeca were homogenised and examined by quantitative bacteriology with media selective for S. hyodysenteriae. There were no differences in any finding due to mouse strain. Group lesion scores over the entire experimental period were significantly higher in mice fed TD (mean total lesion index = 13) than in mice fed CRC (mean total lesion index = 8.8). Lesions were also temporally distributed in a significantly different manner in that they appeared earlier (day 1) and persisted longer in the TD-fed mice in comparison to CRC-fed mice. Furthermore, lesions of equivalent severity from each treatment group presented identical microscopic features. Finally, quantitative bacteriological results indicated that there was no significant difference in the number of cfu of S. hyodysenteriae isolated from mice fed TD and those fed CRC. These results demonstrate that the characteristic severe lesions associated with S. hyodysenteriae infection in mice can occur 1 day after oral challenge in mice fed Teklad diet 85420. Bacteriological results further indicate that the enhancement of lesion formation in this model is not due to any significant effect of the diet on numbers of spirochaetes in the caeca of infected mice.

Animals↗
PubMed2006

Reclassification of Serpulina intermedia and Serpulina murdochii in the genus Brachyspira as Brachyspira intermedia comb. nov. and Brachyspira murdochii comb. nov.

A reassessment was undertaken of published characteristics of the five species of anaerobic intestinal spirochaetes in the genus Brachyspira (Brachyspira aalborgi, Brachyspira alvinipulli, Brachyspira hyodysenteriae, Brachyspira innocens and Brachyspira pilosicoli) and the two species in the genus Serpulina (Serpulina intermedia and Serpulina murdochii). Comparisons were made of published descriptions, including phenotypic properties, grouping by multilocus enzyme electrophoresis, DNA base composition, DNA-DNA relative reassociation values and 16S rRNA gene sequence similarity. On the basis of extensive similarities between all the species, it is proposed to reclassify Serpulina intermedia Stanton et al. 1997 and Serpulina murdochii Stanton et al. 1997 in the genus Brachyspira, as Brachyspira intermedia comb. nov. (type strain ATCC 51140T) and Brachyspira murdochii comb. nov. (type strain ATCC 51284T).

Base Composition↗
PubMed2004

Experimental swine dysentery: comparison between infection models.

The aim of the present study was to develop a reproducible porcine infection model with Brachyspira hyodysenteriae. The influence of different factors was evaluated, namely, age, a diet containing large quantities of soybean meal, housing and administration of cortisol or antacids. Furthermore, the synergistic effect of additional bacteria (Escherichia coli O141, Bacteroides vulgatus or a mixture of Bacteroides fragilis, a field isolate of Bacteroides and Fusobacterium necrophorum) was studied. Experimental infection resulted in an increase in the serum concentrations of the acute-phase proteins serum amyloid A and haptoglobin and the percentages of neutrophils and monocytes. These alterations were specifically related to haemorrhagic diarrhoea. Inoculation combined with feeding of large quantities of soybean meal and group-housing induced swine dysentery in all experimental animals. If the pigs were fed soybean meal, kept in single pens and circulated between the pens, five out of nine developed disease.

Age Factors↗
PubMed2004

Differences in lymphocyte subpopulations and cell counts before and after experimentally induced swine dysentery.

The aim of this study was to examine the levels of circulating leukocytes and lymphocyte subpopulations before and immediately after experimentally induced swine dysentery. Twenty-one healthy crossbred pigs (approximately 22 kg) were orally inoculated with Brachyspira hyodysenteriae. Blood was sampled before inoculation and when clinical signs of swine dysentery occurred. Pigs that remained healthy were sampled when killed. Total and differential white blood cell counts were performed, and lymphocyte subpopulations were analysed using flow cytometry. Following a mean incubation period of 13 days, 12 pigs developed swine dysentery, whereas nine remained healthy throughout the study. Before inoculation, pigs that subsequently developed swine dysentery displayed higher levels of circulating gamma delta T cells (mean +/- se; 30.7 +/- 3.5 %) compared with pigs that remained healthy (14.9 +/- 1.4 %). Sick animals also displayed lower levels of CD8 cells (24.6 +/- 1.5 %), cytotoxic/suppressor T cells (10.9 +/- 1.3 %) and CD4 CD8 T cells (8.1 +/- 1.0 %) than the pigs that remained healthy (34.9 +/- 3.1 %; 17.6 +/- 2.0 %; 13.6 +/- 2.3 %). No difference was observed in leukocyte counts before inoculation. At onset of swine dysentery, there was an increase in monocytes (from 1.5 +/- 0.2 x 10 to 3.8 +/- 0.5 x 10 l) and CD4 CD8 T cells (from 5.8 +/- 0.9 to 8.9 +/- 0.7 %). In conclusion, gamma delta T cells and CD8 cells may be associated with susceptibility to experimentally induced swine dysentery, whereas monocytes and CD4 CD8 T cells appear to be the major responding leukocytes during the disease.

Animals↗
PubMed2004

Brachyspira pilosicoli colonization in experimentally infected mice can be facilitated by dietary manipulation.

The purpose of this study was to determine whether defined dietary manipulations would enhance colonization of mice experimentally challenged with the intestinal spirochaete Brachyspira pilosicoli. Weanling C3H/HeJ mice (n = 48) were fed either a standard balanced mouse diet or a diet supplemented with 50 p.p.m. zinc bacitracin (ZnB), with 50 % (w/w) lactose or with both supplements. Eight mice from each group were challenged orally with a human strain of B. pilosicoli (WesB), whilst four in each group acted as uninfected controls to evaluate the effects of the diets alone. The mice were kept for 40 days following challenge and faeces were collected every 3-4 days and subjected to culture and PCR for B. pilosicoli. Feeding ZnB reduced total volatile fatty acid production in the caecum. Feeding lactose alone doubled the weight of the caecum and its contents compared with control mice, and resulted in a relatively greater production of acetate, but a reduction in propionate and isovalerate production. These effects were negated by the addition of ZnB with the lactose. None of the mice fed the standard diet or receiving ZnB alone became colonized following experimental challenge. One of the mice receiving lactose was culture and PCR positive for B. pilosicoli on one sampling 1 week after inoculation, and one was positive on three samplings taken 20, 25 and 29 days after inoculation. All mice receiving both lactose and ZnB became colonized and remained so, with all samples being positive over the last seven samplings. The colonization was not associated with an end-on attachment of the spirochaete to the epithelial cells of the caecum, but colonized mice developed a specific humoral antibody response to the spirochaete.

Animals↗
PubMed2004

Further characterization of porcine Brachyspira hyodysenteriae isolates with decreased susceptibility to tiamulin.

Brachyspira hyodysenteriae is the causative agent of swine dysentery, a severe diarrhoeal disease in pigs. Few drugs are available to treat the disease, owing to both antimicrobial resistance and withdrawal of drugs authorized for use in pigs. Tiamulin is the drug of choice in many countries, but isolates with decreased susceptibility have recently been reported. The mechanism of tiamulin resistance in B. hyodysenteriae is not known and this facet is essential to understand the dissemination of the trait. To study the resistance epidemiology of B. hyodysenteriae, further characterization of a set of isolates from Germany (n = 16) and the UK (n = 6) with decreased susceptibility to tiamulin was performed. The relatedness between the isolates was studied by comparing PFGE patterns, and the in vitro susceptibility to five other antimicrobials (aivlosin, doxycycline, salinomycin, chloramphenicol and avilamycin) was also determined. For comparison of the antimicrobial-susceptibility pattern, Swedish (n = 20) and British (n = 4) tiamulin-susceptible isolates were tested. The German isolates represented several different PFGE patterns, indicating that tiamulin usage has been sufficient to select clones with decreased tiamulin susceptibility at different farms in Germany. The PFGE pattern for the six British isolates with decreased tiamulin susceptibility was identical to that of the German isolates, and they had a similar antimicrobial-susceptibility pattern, except for resistance to aivlosin, which was only found in a few German isolates. No other co-resistance with tiamulin was found.

Animals↗
PubMed2004

Demonstration of Brachyspira aalborgi lineages 2 and 3 in human colonic biopsies with intestinal spirochaetosis by specific fluorescent in situ hybridization.

Sequences of known 16S rRNA genes, derived from sequence analysis of cloned 16S rDNA, were used to design a specific oligonucleotide probe targeting spirochaetes of Brachyspira aalborgi lineages 2 and 3. The probe was used with fluorescent in situ hybridization to study the involvement of these organisms in human intestinal spirochaetosis. Seventeen human colonic biopsies from Norway and Denmark with intestinal spirochaetosis caused by Brachyspira-like organisms different from the type strain of B. aalborgi (lineage 1) were examined. Application of the probe gave a positive signal in two Norwegian biopsies, whereas the 15 other biopsies were hybridization-negative. The positive reaction visualized the spirochaetes as a fluorescent, 3-5 microm-high fringe on the surface epithelium, extending into the crypts. The study verified the presence of B. aalborgi lineages 2 and 3 and identified the bacteria as an aetiological agent of human intestinal spirochaetosis.

Adult↗
PubMed2004

Extensive intestinal spirochaetosis in pigs challenged with Brachyspira pilosicoli.

A field isolate of Brachyspira pilosicoli, the aetiological agent of porcine intestinal spirochaetosis, was inoculated by stomach tube into six 6-week-old pigs. All animals developed loose to watery faeces and were killed successively on days 8, 14 or 17 post-inoculation (day 17 being the end of the study). Mild mucosal reddening and flecks of pus characterized the gross lesions, while diffuse, catarrhal colitis was revealed microscopically in all animals. Intestinal spirochaetosis with moderate to densely packed end-attached B. pilosicoli organisms was revealed extensively on the mucosal surface of the large intestines by light microscopy and fluorescent in situ hybridization. This study is the first to report extensive intestinal spirochaetosis in pigs challenged with B. pilosicoli.

Animals↗