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PubMed2004

Characterization of spirochetal isolates from arthropods collected in South Moravia, Czech Republic, using fatty acid methyl esters analysis.

Aim of this study was to evaluate cellular fatty acid analysis for characterization of spirochetes. Strains were isolated from arthropods collected in South Moravia, Czech Republic. Fatty acid methyl esters (FAME) profile was determined for five Borrelia burgdorferi sensu lato (s.l.) strains isolated from Ixodes ricinus ticks, one "Spironema culicis" strain recovered from mosquito Culex pipiens and seven spirochetal strains (not identified yet) isolated from mosquitoes and blackflies. Analysis was performed using a gas chromatography column in conjunction with Microbial Identification System Sherlock (MIDI Inc., Newark, DE, USA). Results obtained on the basis of cluster analysis of FAME profiles showed, that the B. burgdorferi sensu lato isolates could be well separated from other spirochetal isolates. We recommended method used in this study as a useful tool for preliminary identification of spirochetes isolated from ticks and dipterans.

Animals↗
PubMed2006

In situ immune response in brain and kidney during early relapsing fever borreliosis.

Characterization of the host immune response during initial pathogenesis of relapsing fever neuroborreliosis would be a key to understanding Borrelia persistence and factors driving the inflammatory process. We analyzed immune cells in brain and kidney with the highly invasive B. crocidurae during the first two weeks of murine infection. In both organs, microglia and/or macrophages predominated while T-cell changes were minimal. Compared to kidney, brain neutrophils infiltrated more rapidly and B-cells were essentially absent. Our results indicate that during early neuroborreliosis, brain defense is comprised primarily of innate immune cells while adaptive immunity plays a minor role.

Animals↗
PubMed2006

A life cycle for Borrelia spirochetes?

Subsequent to Schaudinn and Hoffman's visualization of Treponema pallidum in 1905, many distinguished syphilologists proposed that spirochetes have a life cycle. What is the "essence" of a life cycle? Simply put, life cycles are diverse arrays of life forms, which emerge in an ordered sequence; which are "connected" to one another across primary and secondary hosts, and constitute a cycle with "circular" relationship between hosts. Fecal-oral life cycles and blood-to-blood life cycles are exemplary of host parasite relationships in this realm. The "blood-to-blood" begins and ends with an insect taking a blood "meal". In this operatic scenario, a "blood-less" insect functions simultaneously as a hypodermic needle and as an incubator for some of the infectious components. The initial phase is inside the body fluid compartment of an insect. The second phase is in the blood or body fluid of a warm-blooded mammal. Third, is the phase inside the cell of a mammalian host. And a final portion of the "life" marked by "death" of the parasitized mammalian cells and the release of infectious parasites which return to the "warm" blood where the "cold blooded" vector again takes a blood meal. The cycle then begins again. In each phase of a blood to blood life cycle, the infectious agent changes its shape. Blood phase "profiles" look different from "tissue phase" profiles. Some of the tissue phase profiles may be "invisible". Borrelia spirochetes offer an excellent example of a life cycle, by virtue of the insect vector to mammalian "piece", the blood and intracellular residence "pieces" and the morphologic diversity "piece". Stereotypes of what a spirochete "should " look like, have actually produced a state of "perseveration" in spirochetal pathobiology. We have been "stuck" like a broken record, on the corkscrew form, and have failed to see the rest of the life cycle. Cystic, granular, and cell wall deficient spirochetal profiles, which were well known in the 19th and 20th centuries by such titans as Schaudinn, Hoffman, Noguchi, Delamater, Steiner, and Mattman, have been repudiated by professional microbiologists, and by pathologists who practice and who confer the status of 21st century truths in microbiology matters. Proper microscopic study, as is required by Dr. Robert Koch's second postulate, for establishing links between microbes and disease, presupposes that the microscopist be aware of the complete array of morphologic repertoires of the alleged pathogen. (Morphologies, which are herein introduced.).

Animals↗
PubMed2006

Spirochetal cyst forms in neurodegenerative disorders,...hiding in plain sight.

Here is proposed a hypothesis that a completely unsuspected biology exists for pathogenic spirochetes, namely that the cystic spirochetal forms (long thought to be static and resting or just a dormant cohort) actually are capable of killing mammalian host cells. At least two "lethal" scenarios are proposed; first, the host cell destruction from the "inside out" by small caliber cystic forms invading the host cell cytoplasm, and second host cell destruction by engulfment of entire host cells by large caliber cystic spirochetal forms. Conventional thinking about spirochetal cyst forms is divided between two polar spheres of influence; one a majority community that completely denies the existence of spirochetal cyst forms, and a second group of academically persecuted individuals who accepts the precepts of such antebellum scientists as Schaudinn, Hoffman, Dutton, Levaditi, Balfour, Fantham, Noguchi, McDonough, Hindle, Steiner, Ingraham, Coutts, Hampp, Warthin, Ovcinnikov, and Delamater. Microscopic images of cystic spirochetes are difficult to ignore, but as has been the case in this century, academic "endowments" have nearly expunged all cystic spirochetal image data from the current textbook versions of what is the truth about the spirochetaceae. If the image database from the last century is obliterated; many opportunities to diagnose will be lost. Variously sized cystic spirochetal profiles within diseased nerve cells explain the following structures: Lewy body of Parkinson's disease, Pick body, ALS spherical body, Alzheimer plaque. Borrelia infection is therefore a unifying concept to explain diverse neurodegenerative diseases, based not entirely on a corkscrew shaped profile in diseased tissue, but based on small, medium and large caliber rounded cystic profiles derived from pathogenic spirochetes which are..."hiding in plain sight".

Borrelia Infections↗
PubMed2006

Induction of differential immune reactivity to members of the flora of gnotobiotic mice following colonization with Helicobacter bilis or Brachyspira hyodysenteriae.

Aberrant host immune responses to bacterial components of the resident microflora may initiate and perpetuate gastrointestinal inflammation. To investigate how microbial perturbation promotes host immunological responsiveness to commensal bacteria and contributes to the development of typhlocolitis, we selectively colonized defined (altered Schaedler) flora C3H mice with either Helicobacter bilis or Brachyspira hyodysenteriae. Following selective colonization, tissues were analyzed for gross/histopathologic lesions and bacterial antigen-specific B- and T-cell responses. Gnotobiotic mice colonized with H. bilis or B. hyodysenteriae developed typhlocolitis of varying severity, with the most severe gross and histopathogical lesions observed in B. hyodysenteriae-colonized mice. Antigen-specific IgG1 and IgG2a responses to the resident microflora were increased in both H. bilis-and B. hyodysenteriae-colonized mice. The greater antibody responses were associated with less severe cecal inflammation in H. bilis-colonized mice. Altered Schaedler flora (ASF)-stimulated mesenteric lymphocytes from B. hyodysenteriae-colonized mice produced higher levels of interferon-gamma and interleukin (IL)-4 than did lymphocytes from H. bilis-colonized mice. However, ASF-stimulated mesenteric and splenic lymphocytes from both H. bilis and B. hyodysenteriae-colonized mice secreted higher amounts of IL-10 compared to similarly stimulated lymphocytes recovered from control mice. These results indicate that microbial perturbation may induce differential immune responses to nonpathogenic resident bacteria that can lead to intestinal inflammation.

Animals↗
PubMed2006

Comparison of culture and biochemical tests with PCR for detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli.

Traditional culture and biochemical tests (CBT) were compared with PCR for sensitivity and detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli in seeded faeces and clinical samples from diarrhoeic pigs. A duplex PCR system was developed based on primers detecting the tlyA-gene of B. hyodysenteriae and the 16S rRNA-gene of B. pilosicoli. Sensitivities for the PCR system were determined on seeded faeces, using DNA that had been recovered from primary cultures or extracted directly from faeces. Compared to CBT, PCR applied to DNA extracted directly from faeces lowered the sensitivity by a factor of 1000 to 10,000. B. hyodysenteriae and B. pilosicoli detection was compared for CBT and PCR using 200 clinical samples. CBT detected more B. hyodysenteriae isolates in the clinical samples than PCR, but fewer B. pilosicoli positive samples. An atypical strongly haemolytic isolate was detected only by CBT.

Animals↗
PubMed2005

Tiamulin resistance in porcine Brachyspira pilosicoli isolates.

There are few studies on antimicrobial susceptibility of Brachyspira pilosicoli, therefore this study was performed to investigate the situation among isolates from pigs. The tiamulin and tylosin susceptibility was determined by broth dilution for 93 and 86 porcine B. pilosicoli isolates, respectively. The isolates came from clinical samples taken in Swedish pig herds during the years 2002 and 2003. The tylosin minimal inhibitory concentration (MIC) was >16 microg/ml for 50% (n=43) of the isolates tested. A tiamulin MIC >2 microg/ml was obtained for 14% (n=13) of the isolates and these were also tested against doxycycline, salinomycin, valnemulin, lincomycin and aivlosin. For these isolates the susceptibility to salinomycin and doxycycline was high but the MICs for aivlosin varied. The relationship between the 13 tiamulin resistant isolates was analyzed by pulsed-field gel electrophoresis (PFGE). Among the 13 isolates 10 different PFGE patterns were identified.

Animals↗
PubMed2006

Consecutive pathological and immunological alterations during experimentally induced swine dysentery - a study performed by repeated endoscopy and biopsy samplings through an intestinal cannula.

The development of intestinal lesions after inoculation with Brachyspira hyodysenteriae was followed by repeated endoscopy and biopsy sampling through a caecal cannula. Seven eight-week-old pigs were cannulated and inoculated, two were cannulated but not inoculated, and two pigs were inoculated but not cannulated. Endoscopy, biopsy, and blood sampling to determine SAA (serum amyloid A), haptoglobin, cortisol, and WBC counts were performed at scheduled time-points. At the third day of disease, endoscopy showed a hyperaemic, perturbed mucosa and excessive amount of mucus. Histologically, crypt hyperplasia, depletion of goblet cell mucus, and erosions were noted. Simultaneously, elevated acute phase proteins and circulating monocytes, and decreased number of intraepithelial CD3(+) cells were observed. After five days the pigs recovered. Intestinal lesions were demarcated and interspersed among apparently normal mucosa and blood parameters returned to initial values. Endoscopy through an intestinal cannula made it possible to follow the development of intestinal alterations in vivo and describe the sequential events during the course of swine dysentery. The number of animals used in a study could thus be minimised and the precision of the experiment increased.

Animals↗
PubMed2006

Blood concentrations of amino acids, glucose and lactate during experimental swine dysentery.

The aim of this study was to examine blood concentrations of amino acids, glucose and lactate in association with experimental swine dysentery. Ten pigs (approximately 23kg) were orally inoculated with Brachyspira hyodysenteriae. Eight animals developed muco-haemorrhagic diarrhoea with impaired general appearance, changes in white blood cell counts and increased levels of the acute phase protein Serum Amyolid A. Blood samples were taken before inoculation, during the incubation period, during clinical signs of dysentery and during recovery. Neither plasma glucose nor lactate concentrations changed during the course of swine dysentery, but the serum concentrations of gluconeogenic non-essential amino acids decreased during dysentery. This was mainly due to decreases in alanine, glutamine, serine and tyrosine. Lysine increased during dysentery and at the beginning of the recovery period, and leucine increased during recovery. Glutamine, alanine and tyrosine levels show negative correlations with the numbers of neutrophils and monocytes. In conclusion, swine dysentery altered the blood concentrations of amino acids, but not of glucose or lactate.

Amino Acids↗
PubMed2005

Bovine digital dermatitis and severe virulent ovine foot rot: a common spirochaetal pathogenesis.

A potential pathological role for spirochaetes in bovine digital dermatitis (bovine DD) and severe virulent ovine foot rot (SVOFR) has been considered and a treponeme isolate obtained from each disease in the UK. In this work, we have investigated the hypothesis that the two diseases may have a shared (common) spirochaetal aetiology. Experiments were designed to identify serological similarities and differences between the two spirochaetes; an enzyme-linked immunosorbent assay (ELISA) was developed to detect anti-treponeme antibodies in the sera of cows and sheep against the two-treponeme isolates. Sera were further tested for antigen reactivity by Western blotting. Cattle and sheep with bovine DD and SVOFR, respectively, had increased seropositivity rates to both treponeme isolates, with different patterns of reactivity between farms. In some cattle herds, significant correlations were shown between antibodies to bovine DD treponemes and SVOFR treponemes (P<0.001). In other herds, there was no apparent cross reaction, suggesting the presence of more than one treponeme in bovine DD on some farms. There was no significant correlation between the two treponeme isolates when ELISA-tested against 58 sheep sera from SVOFR cases (P>0.05); sheep showed strong evidence of reactivity to one or the other treponeme antigens, but never to both. Western blotting against both treponeme antigens showed that they frequently displayed different antigen epitopes, although some minor bands were common to both organisms. The data suggest that there are a number of spirochaetes in UK farms, which could be involved in the pathogenesis of either bovine DD or SVOFR.

Animals↗
PubMed2005

Assessment of diagnostics and antimicrobial susceptibility testing of Brachyspira species using a ring test.

There is no ring test for quality assessment available in Europe for diagnostics and antimicrobial susceptibility testing of the fastidious, anaerobic bacteria of the genus Brachyspira. Therefore, an international ring test for Brachyspira spp. was performed once a year during 2002-2004. Two sets of coded samples were prepared and distributed on each occasion. One set comprised six swabs dipped in pig faeces spiked with Brachyspira spp. intended for diagnostics. The other set comprised two pure strains intended only for susceptibility testing. All methods used were in-house methods. The species used were Brachyspira hyodysenteriae, Brachyspira pilosicoli, Brachyspira innocens, Brachyspira murdochii and Brachyspira intermedia. In most cases, the correct Brachyspira spp. were detected. However, the results showed that Brachyspira spp. could be difficult to identify, especially if two Brachyspira spp. were mixed or if the concentration of Brachyspira in faeces was low. Additionally, some laboratories reported Brachyspira growth in control samples that were not seeded with any spirochaetes. The lowest detection level was 10(2) bacteria/ml faeces for both B. hyodysenteriae and B. pilosicoli. The susceptibility tests performed showed that disc diffusion was not recommendable for Brachyspira spp. Extended antimicrobial dilution series gave most congruent results. The diversity of the results highlights the importance of ring tests for a high quality of diagnostics and antimicrobial susceptibility tests for Brachyspira spp. This is the first ring test described for Brachyspira spp.

Animals↗
PubMed2005

SmpB: a novel outer membrane protein present in some Brachyspira hyodysenteriae strains.

A novel outer membrane protein-encoding gene was identified in Brachyspira hyodysenteriae. The predicted protein, SmpB, was encoded by a gene that contains regions of identity with that encoding the previously identified lipoprotein SmpA. However, the majority of the reading frame encoding SmpA and SmpB share no detectable similarity. Analysis of several strains revealed that B. hyodysenteriae harbours either smpA or the newly identified gene smpB, but not both. smpB encodes for a slightly larger protein than smpA, 17.6 and 16.8 kDa, respectively. The predicted proteins share an identical leader sequence and the first 10 amino acids of the mature protein, however, the remainder of the predicted protein sequence shows no similarity. It is hypothesised that smpA and smpB are present on the same area of the chromosome. The proteins are antigenically unique, as antisera raised against a strain of B. hyodysenteriae that expresses SmpA cannot detect SmpB and vice versa. Although the presence of an identical leader peptide suggests identical localisation of SmpA and SmpB, it is not known if the two predicted proteins share similar function.

Animals↗
PubMed2000

Polymerase chain reaction for the detection of flaA-1 genes of oral spirochaetes in human advanced periodontal pockets.

A novel group of oral spirochaetes that were specifically labeled by H9-2, a monoclonal antibody against the 37-kDa endoflagellar sheath protein of Treponema pallidum, have earlier been demonstrated in periodontitis-associated plaque samples; two cultivable oral spirochaetes T. vincentii and T. medium displayed cross-reactivity with that monoclonal, but the molecular basis for this cross-reactivity is not yet defined. Here, the flaA-1 genes which encode the 37-kDa sheath protein from T. vincentii and the oral spirochaetes present in samples from advanced periodontitis were examined. A 856 bp fragment of the flaA-1 gene was amplified in T. pallidum and T. vincentii. The same-size polymerase chain reaction fragments were also amplified in two clinical samples from patients with advanced periodontitis but not from samples of healthy plaque. The sequences of the flaA-1 genes of the oral spirochaetes detected in human periodontal plaque were closely similar to those of T. pallidum and T. vincentii, but neither of these two organisms could be detected in these samples using rRNA-specific primers. The identity of the flaA-1 positive spirochaetes associated with periodontitis remains to be determined.

Antibodies, Monoclonal↗
PubMed1998

Evaluation of day-old specific pathogen-free chicks as an experimental model for pathogenicity testing of intestinal spirochaete species.

Specific pathogen-free chicks aged 1 day were challenged per os with strains of five different species of intestinal spirochaete originally isolated from pigs or human beings. A virulent strain of Serpulina hyodysenteriae (WA 15) colonized chicks, causing retarded growth rate and histological changes, including caecal atrophy, epithelial and goblet cell hyperplasia, and crypt elongation. A further strain of S. hyodysenteriae (SA3), which was apparently avirulent for pigs, and a strain of Serpulina intermedia (889) colonized fewer chicks, caused less severe lesions and did not significantly depress growth rate. Strains of Serpulina murdochii and Brachyspira aalborgi failed to colonize or cause histological changes. Four strains of Serpulina pilosicoli (Kar, Rosie-2299 and GAP 401, isolated from human beings, and 3295, isolated from a pig) colonized chicks, and large numbers showed polar attachment to the caecal epithelium; all strains, apart from Rosie-2299, caused watery diarrhoea and wet litter, but did not significantly retard growth. Variation both in the degree of spirochaetal attachment and the resulting development of lesions was observed between S. pilosicoli strains as well as between individual chicks infected with the same strain. The study indicated that chicks may be useful in studying the pathogenicity of strains of S. hyodysenteriae, S. intermedia and S. pilosicoli.

Animals↗