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PubMed1997

Immunoblot reactivity of polyclonal and monoclonal antibodies with periplasmic flagellar proteins FlaA1 and FlaB of porcine Serpulina species.

The periplasmic-flagellum (PF) proteins of Triton X-100-soluble and Triton X-100-insoluble sodium dodecyl sulfate-treated fractions from reference and field strains of Serpulina hyodysenteriae, Serpulina innocens, and Serpulina pilosicoli were characterized by Western blotting with a rabbit polyclonal antibody (PAb) specific for the 44-kDa PF sheath protein of S. hyodysenteriae (Z. Li, F. Dumas, D. Dubreuil, and M. Jacques, J. Bacteriol. 175:8000-8007, 1993) and a murine monoclonal antibody (MAb), designated 7G2, specific for the PF core FlaB proteins of S. hyodysenteriae. The MAb 7G2 reacted with a conserved epitope present in the 37-, 34-, and 32-kDa PF core FlaB proteins of all Serpulina species. This suggested that the core FlaB proteins are conserved among porcine Serpulina species. An immunoreactive band of approximately 44 kDa was present with all S. hyodysenteriae, S. innocens, and S. pilosicoli strains that were reacted with the PAb. The specificities of the PAb and the MAb for the FlaA1 and FlaB proteins of Serpulina species were confirmed by N-terminal amino acid sequencing of 44- and 37-kDa proteins, respectively, of S. hyodysenteriae and S. pilosicoli. Results from this study provide further evidence that the 44-kDa protein FlaA1 and the 37-, 34-, and 32-kDa FlaB proteins are conserved among porcine Serpulina species.

Amino Acid Sequence↗
PubMed2006

Borrelia burgdorferi spirochetes that harbor only a portion of the lp28-1 plasmid elicit antibody responses detectable with the C6 test for Lyme disease.

Detection of antibody to C6, a peptide that reproduces the sequence of the sixth invariable region within the central domain of the VlsE protein of Borrelia burgdorferi, is used currently for the serologic diagnosis of Lyme disease in humans. B. burgdorferi isolates taken from infected humans can be categorized into specific genetic subtypes (designated RST1, -2, and -3) by restriction fragment length polymorphisms in the 16S to 23S rRNA spacer sequence. Many of these, usually categorized as RST2, retain only segments of the linear plasmid lp28-1, which encodes VlsE. The VlsE genetic region is retained, but altered expression of this molecule could affect diagnosis by the C6 enzyme-linked immunosorbent assay (ELISA). Serum samples from patients infected with each of the three genotypes and from mice infected with three RST2 isolates were tested with the C6 ELISA. Such isolates elicited marked C6 responses in infected mice. The sensitivity of C6 antibody detection in patients infected with RST2 spirochetes was statistically indistinguishable from detection of RST1 and RST3 infections. These findings demonstrate that diagnosis by C6 ELISA remains effective for infection with all B. burgdorferi genotypes, including those with incomplete lp28-1 plasmids.

Animals↗
PubMed2006

Differential expression of the Bhmp39 major outer membrane proteins of Brachyspira hyodysenteriae.

The enteric, anaerobic spirochete Brachyspira hyodysenteriae is the causative agent of swine dysentery, a severe mucohemorrhagic diarrheal disease of pigs that has economic significance in every major pork-producing country. Recent investigation into potential vaccine candidates has focused on the outer membrane proteins of B. hyodysenteriae. Bhmp39 (formerly Vsp39) is the most abundant surface-exposed outer membrane protein of B. hyodysenteriae; its predicted gene sequence has previously been shown to share sequence similarity to eight genes divided evenly between two paralogous loci. The peptide sequence suggested that Bhmp39 is encoded by one of these genes, bhmp39h. The biological significance of maintaining eight homologous bhmp39 genes is unclear, though it has been proposed that this may play a role in antigenic variation. In this study, real-time, reverse transcription-PCR was used to demonstrate that bhmp39f and bhmp39h were the transcripts most abundantly expressed by B. hyodysenteriae strain B204 cultured under in vitro growth conditions. Mass spectrometry data of the purified 39-kDa membrane protein showed that both Bhmp39f and Bhmp39h were present. Northern blot analysis across predicted Rho-independent terminators demonstrated that the genes of the bhmp39efgh locus result in monocistronic transcripts.

Amino Acid Sequence↗
PubMed1982

In vitro lymphocyte blastogenic responses and titers of humoral antibodies from periodontitis patients to oral spirochete isolates.

Humoral antibodies and in vitro blastogenic responses of patients with moderate to advanced periodontitis and of orally healthy (control) individuals to 11 spirochetes were assayed. Eight isolates of Treponema denticola, two isolates of T. vincentii, and one isolate of T. phagedenis were grown anaerobically, sonicated, and centrifuged to remove cellular debris. The blastogenic responses of peripheral blood lymphocytes were measured by [3H]-thymidine uptake. Antibody titers (immunoglobulins A, G, and M) to the spirochetes were measured with the micro-enzyme-linked immunosorbent assay. Dark-field microscopic enumeration of spirochetes was made from subgingival plaque. Spirochetes comprised 20% of the total number of microorganisms seen in plaque from the patients, whereas no spirochetes were seen in plaque from the controls. The blastogenic responses to the spirochetes were not statistically different (P greater than 0.05) between patients and controls. The immunoglobulin G and M antibody titers of the patients were generally lower than the titers of the controls, but the differences were rarely statistically significant. In contrast, the patients possessed significantly higher immunoglobulin A titers to seven spirochetes (T. denticola). No correlation was found between the percentage of spirochetes in the plaque sample and the blastogenic responses or titers of antibodies from an individual to the 11 spirochetes.

Adult↗
PubMed1982

Common antigens of Treponema denticola: chemical, physical, and serological characterization.

A sodium deoxycholate-ethanol extractable antigen (DES-Ag) was obtained from four serotypes of Treponema denticola and characterized by chemical, physical, and serological procedures. The cross-reactivity of these antigens was demonstrated by indirect microhemagglutination, immunodiffusion, and immunoelectrophoresis with hyperimmune rabbit antiserum against each of the T. denticola serotypes. Antiserum against two nonoral treponemes, T. phagedenis biotype Reieter and T. pallidum Nichols strain, did not cross-react with the DES-Ag of T. denticola. Chemical analysis of the DES-Ag indicated that proteins (84%) and hexoses (12%) accounted for 96% of the total dry weight of the antigen. Trace amounts of N-acetylglucosamine (0.6%) were also detected. Fatty acids, including palmitic, oleic, and stearic, were identified by gas-liquid chromatography. Polyacrylamide gel electrophoresis of the DES-Ag of each serotype revealed the presence of two bands. The molecular weights of the bands were estimated to be 58,000 and 31,000 by comparing the electrophoretic mobility of the DES-Ag to that of five protein standards of known molecular weights. Although only a single precipitin line was observed by immunodiffusion when each antigen was reacted against its homologous antiserum, two precipitin lines were evident by immunoelectrophoresis. Antiserum against the DES-Ag of T. denticola was shown to agglutinate whole cells of the homologous serotype. Adsorption of this anti-DES-Ag serum with whole cells of T. denticola resulted in the elimination of precipitating antibodies to the DES-Ag by immunodiffusion. It is concluded that the DES-Ag is a component of the outer sheath of T. denticola.

Animals↗
PubMed1984

Variation in a major surface protein of Lyme disease spirochetes.

Two monoclonal antibodies (H6831 and H5TS) differed in their indirect immunofluorescence reactivity when tested against 14 strains of Lyme disease spirochetes. Strains were bound by both antibodies, by H6831 or H5TS alone, or by neither. Western blot and immunoprecipitation studies revealed that the determinants of both antibodies were associated with abundant proteins with apparent subunit molecular weights of ca. 34,000 (34K-range proteins). The following results indicated that the 34K-range proteins were exposed on the surface of the spirochetes. (i) Antibody H6831 agglutinated the spirochetes; (ii) immune electron microscopy showed that the H6831 determinant was associated with the outer membrane; (iii) radiolabeled H6831 bound to live organisms; and (iv) proteases effectively removed the 34K-range proteins from intact cells. With their demonstrated variability and exposure on the surface, the 34K-range proteins may contribute to the serotype specificity of Lyme disease spirochetes.

Agglutination Tests↗
PubMed1984

Experimental Lyme disease in rabbits: spirochetes found in erythema migrans and blood.

In attempts to produce experimental Lyme disease, 33 rabbits were inoculated with Lyme spirochetes by tick feeding or from tick organ homogenates or cultures. Two rabbits developed erythema chronicum migrans at the site of inoculation, in one instance 2 days after injection of a tick organ homogenate and in the other instance, 17 days after feeding of infected Ixodes dammini ticks. Spirochetes were seen in skin biopsy specimens of the second lesion with Warthin-Starry and immunoperoxidase stains. Spirochetes were also recovered from blood cultures of two additional rabbits 2 weeks post-inoculation. These findings are characteristic of early Lyme disease in humans and give additional support for the spirochetal etiology of Lyme disease.

Animals↗
PubMed1985

Bacteroides endodontalis and other black-pigmented Bacteroides species in odontogenic abscesses.

Twenty-eight odontogenic abscesses were examined for the presence of black-pigmented Bacteroides spp. Of the 28 samples, 26 were found to contain one or more species of black-pigmented Bacteroides. Abscesses were divided into three categories according to the tissue of origin: endodontal, periodontal, and pericoronal. Four abscesses which developed after extraction were also examined. It was found that Bacteroides endodontalis, a newly described species of asaccharolytic black-pigmented Bacteroides, was isolated almost exclusively from periapical abscesses of endodontal origin. B. intermedius proved to be the most frequently isolated species in all of the samples. B. gingivalis was present in all of the periodontal abscesses studied, as well as in two endodontal abscesses. B. melaninogenicus was recovered once from a pericoronal abscess. Precautions for the isolation of B. endodontalis are discussed.

Adolescent↗
PubMed1989

Comparative analysis of the genomes of intestinal spirochetes of human and animal origin.

The aim of the present work was to compare the genomes of 21 strains of intestinal spirochetes, which were isolated from patients suffering intestinal disorders, with those of Treponema hyodysenteriae (strain P18), the known etiological agent of swine dysentery (bloody scours), and of a nonpathogenic strain (M1) of Treponema innocens. The percent guanine-plus-cytosine value of the 23 DNAs was found to be 25.5 to 30.1, as determined by a double-labeling procedure based on nick-translation by DNA polymerase I. The genome size of two spirochetal strains, of human and porcine origin, was found to be similar (4 x 10(6) base pairs) and close to that of the reference bacterium Escherichia coli (4.2 x 10(6) base pairs). Restriction analysis showed the presence of two modified bases in spirochetal DNA. Methyladenine was present in the GATC sequence of DNA from 15 spirochetes of human origin, and methylcytosine was present in several sequences occurring in all strains. The DNA of T. hyodysenteriae displayed a 30 to 100% homology with respect to that of 21 spirochetes from humans, thus suggesting the occurrence of a genetic heterogeneity in the latter group. These data indicate that the intestinal spirochetes analyzed in the present work are related; hence there is a possibility of domestic animals being reservoirs of microorganisms pathogenic for humans. A classification of intestinal treponemes into subgroups has been proposed on the basis of restriction analysis and hybridization experiments.

Animals↗
PubMed1994

Chemotactic response to mucin by Serpulina hyodysenteriae and other porcine spirochetes: potential role in intestinal colonization.

Chemotaxis of porcine spirochetes towards a variety of mucins was measured quantitatively by a capillary method. A chemotaxis buffer consisting of 0.01 M potassium phosphate buffer (pH 7.0) and 0.2 mM L-cysteine hydrochloride was necessary for chemotaxis of spirochetes. The optimum incubation time and incubation temperature were 1 h and 40 degrees C, respectively. The mucin concentration also affected the chemotaxis observed, and a concentration of 1% (wt/vol) was near the optimum. Virulent Serpulina hyodysenteriae strains were chemotactic towards 1% (wt/vol) hog gastric mucin and 1% (wt/vol) porcine colonic mucin but not towards 1% (wt/vol) bovine submaxillary mucin. Virulent S. hyodysenteriae strains were significantly more chemotactic than avirulent strains of S. hyodysenteriae (SA3 and VS1), Serpulina intermedius, and Serpulina innocens. Other spirochetes belonging to the proposed group of spirochetes Anguillina coli were also not chemotactic. Pathogenicity of S. hyodysenteriae strains that cause swine dysentery may, in part, be attributed to their attraction to porcine intestinal mucus.

Animals↗
PubMed1995

Pathogenicity of human and porcine intestinal spirochetes in one-day-old specific-pathogen-free chicks: an animal model of intestinal spirochetosis.

One-day-old chicks were infected orally with two strains of weakly hemolytic spirochetes isolated from a human and a pig with intestinal spirochetosis. These spirochetosis both colonized birds, attached end-on to their cecal enterocytes, induced watery diarrhea, and significantly depressed growth rates. Cultures of Serpulina innocens failed to colonize the chicks.

Animals↗
PubMed1996

Dual flaA1 flaB1 mutant of Serpulina hyodysenteriae expressing periplasmic flagella is severely attenuated in a murine model of swine dysentery.

The motility imparted by the periplasmic flagella (PF) of Serpulina hyodysenteriae is thought to play a pivotal role in the enteropathogenicity of this spirochete. The complex PF are composed of multiple class A and class B polypeptides. Isogenic strains containing specifically disrupted flaAl or flaB1 alleles remain capable of expressing PF, although such mutants display aberrant motility in vitro. To further examine the role that these proteins play in the maintenance of periplasmic flagellar structural integrity, motility, and fitness for intestinal colonization, we constructed a novel strain of S. hyodysenteriae which is deficient in both FlaA1 and FlaB1. To facilitate construction of this strain, a chloramphenicol gene cassette, with general application as a selectable marker in prokaryotes, was developed. The cloned flaAl and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and kanamycin gene cassettes, respectively. The inactivated flagellar genes were introduced into S. hyodysenteriae, and allelic exchange at the targeted chromosomal flaA1 and flaB1 loci was verified by PCR analysis. Immunoblots or cell lysates with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath and core proteins in this dual flagellar mutant. These mutations selectively abolished the expression of the targeted genes without affecting the synthesis of other immunologically related FlaB proteins. The resulting flaA1 flaB1 mutant exhibited altered motility in vitro. Surprisingly, it was capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy. The virulence of this strain was assessed in a murine model of swine dysentery by determining the incidence of cecal lesions and the persistence of S. hyodysenteriae in the gut. Mice challenged with the wild-type strain or a passage control strain showed a dose-related response to the challenge organism. The dual flagellar mutant was severely attenuated in murine challenge experiments, suggesting that the FlaA1 and FlaB1 proteins are dispensable for flagellar assembly but critical for normal flagellar function and colonization of mucosal surfaces of the gastrointestinal tract. This strain represents the first spirochete engineered to contain specifically defined mutations in more than one genetic locus.

Animals↗
PubMed1996

Experimental infection of newly weaned pigs with human and porcine strains of Serpulina pilosicoli.

Cultures of Serpulina pilosicoli 95/1000, isolated from a pig with porcine intestinal spirochetosis (PIS), and S. pilosicoli WesB, isolated from an Aboriginal child with diarrhea, were used to infect 5-week-old newly weaned pigs. Four of 12 pigs infected with strain 95/1000 and 2 of 12 pigs infected with strain WesB became colonized and developed watery, mucoid diarrhea within 2 to 11 days postinfection. Affected pigs all had moderate subacute mucosal colitis, with gross and histological changes similar to those previously reported in both natural and experimentally induced cases of PIS. Silver-stained histological sections of the colon and cecum from affected pigs demonstrated spirochetes within dilated intestinal crypts, where they were associated with neutrophilic exocytosis and mucus secretion. Sections from one pig infected with strain 95/1000 showed large numbers of spirochetes attached by one end to the colonic epithelium, a feature consistent with PIS. This study confirms the role of S. pilosicoli in the etiology of PIS and provides evidence that S. pilosicoli strains of human origin have pathogenic potential in an animal model.

Animals↗
PubMed1997

Experimental infection of C3H mice with avian, porcine, or human isolates of Serpulina pilosicoli.

C3H/HeJ (lps(d)/lps(d)) and C3H/HeOuJ (lps(n)/lps(n)) mice were infected via gastric intubation with avian, porcine, or human isolates of weakly hemolytic spirochetes classified as Serpulina pilosicoli. Upon histopathological examination of cecal tissue from mice infected with avian or porcine isolates, colonization of spirochetes attached end-on to the apical surface of enterocytes was observed. There were no apparent differences in severity of cecal lesions between the lipopolysaccharide (LPS)-responsive (C3H/HeOuJ) and LPS-hyporesponsive (C3H/HeJ) mouse strains infected with these isolates. Transmission electron microscopy showed spirochetes invaginated into the host cell membrane with resultant effacement of microvilli and loss of the glycocalyx. End-on attachment of the human isolate S. jonesii was not observed in the present studies, although weakly hemolytic spirochetes were reisolated from mice infected with S. jonesii. Moreover, results of Western immunoblot experiments showed mice developed serum antibody responses to the S. pilosicoli isolates examined. Thus, the present results indicate that specific isolates of S. pilosicoli can colonize mice and exhibit end-on attachment to cecal enterocytes.

Animals↗
PubMed1999

Strain variation in glycosaminoglycan recognition influences cell-type-specific binding by lyme disease spirochetes.

Lyme disease, a chronic multisystemic disorder that can affect the skin, heart, joints, and nervous system is caused by Borrelia burgdorferi sensu lato. Lyme disease spirochetes were previously shown to bind glycosaminoglycans (GAGs). In the current study, the GAG-binding properties of eight Lyme disease strains were determined. Binding by two high-passage HB19 derivatives to Vero cells could not be inhibited by enzymatic removal of GAGs or by the addition of exogenous GAG. The other six strains, which included a different high-passage HB19 derivative (HB19 clone 1), were shown to recognize both heparan sulfate and dermatan sulfate in cell-binding assays, but the relative efficiency of binding to these two GAGs varied among the strains. Strains N40, CA20-2A, and PBi bound predominantly to heparan sulfate, PBo bound both heparan sulfate and dermatan sulfate roughly equally, and VS461 and HB19 clone 1 recognized primarily dermatan sulfate. Cell binding by strain HB19 clone 1 was inhibited better by exogenous dermatan sulfate than by heparin, whereas heparin was the better inhibitor of binding by strain N40. The GAG-binding preference of a Lyme disease strain was reflected in its cell-type-specific binding. Strains that recognized predominantly heparan sulfate bound efficiently to both C6 glioma cells and EA-Hy926 cells, whereas strains that recognized predominantly dermatan sulfate bound well only to the glial cells. The effect of lyase treatment of these cells on bacterial binding was consistent with the model that cell-type-specific binding was a reflection of the GAG-binding preference. We conclude that the GAG-binding preference varies with the strain of Lyme disease spirochete and that this variation influences cell-type-specific binding in vitro.

Animals↗
PubMed2000

Identification of a Treponema denticola OppA homologue that binds host proteins present in the subgingival environment.

Proteins secreted or exported by Treponema denticola have been implicated as mediators of specific interactions between the spirochete and subgingival tissues in periodontal diseases. However, limited information is available on the ability of this peptidolytic organism to bind or transport soluble peptides present in the subgingival environment. A prominent 70-kDa protein was isolated from surface extracts of T. denticola ATCC 35405. A clone expressing a portion of the protein was identified in an Escherichia coli expression library of T. denticola DNA. DNA sequence analysis showed that the cloned gene encoded a peptide homologous to OppA, the solute binding protein of an ATP-binding cassette-type peptide transporter involved in peptide uptake and environmental signaling in a wide range of bacteria. Genes encoding OppB, -C, -D, and -F were identified directly downstream of oppA in T. denticola. OppA was present in representative strains of T. denticola and in Treponema vincentii but was not detected in Treponema pectinovorum or Treponema socranskii. Immunogold electron microscopy suggested that OppA was accessible to proteins at the surface of the spirochete. Native OppA bound soluble plasminogen and fibronectin but did not bind to immobilized substrates or epithelial cells. A T. denticola oppA mutant bound reduced amounts of soluble plasminogen, and plasminogen binding to the parent strain was inhibited by the lysine analog epsilon-aminocaproic acid. Binding of soluble host proteins by OppA may be important both for spirochete-host interactions in the subgingival environment and for uptake of peptide nutrients.

Alleles↗
PubMed2000

Cloning and DNA sequence analysis of an immunogenic glucose-galactose MglB lipoprotein homologue from Brachyspira pilosicoli, the agent of colonic spirochetosis.

Colonic spirochetosis (CS) is a newly emerging infectious disease of humans and animals caused by the pathogenic spirochete Brachyspira (formerly Serpulina) pilosicoli. The purpose of this study was to characterize an antigen that was recognized by antibodies present in sera of challenge-exposed pigs. The gene encoding the antigen was identified by screening a plasmid library of human B. pilosicoli strain SP16 (ATCC 49776) genomic DNA with hyperimmune and convalescent swine sera. The predicted amino acid sequence encoded by the cloned B. pilosicoli gene had a high degree of similarity and identity to glucose-galactose MglB lipoprotein. Located 106 bp downstream of the putative mglB gene was a 3'-truncated open reading frame with 73.8% similarity and 66.3% identity to mglA of Escherichia coli, suggesting a gene arrangement within an operon which is similar to those of other bacteria. A single copy of the gene was present in B. pilosicoli, and homologous sequences were widely conserved among porcine intestinal spirochetes Serpulina intermedia, Brachyspira innocens, Brachyspira murdochii, and the avian Brachyspira alvinipulli, but not in porcine Brachyspira hyodysenteriae, human Brachyspira aalborgi, and porcine Treponema succinifaciens. The deduced molecular weight of the mature MglB lipoprotein was consistent with expression by the cloned gene of a polypeptide with an apparent molecular weight of 36,000, as determined by Western blot analysis and [(3)H]palmitate labeling. Because mucin is the principal constituent of the colonic mucus gel and consists of glycoproteins that can serve as the substrate for growth and chemotaxis of B. pilosicoli in vitro, a role for MglB in mucosal localization of the spirochete appears consistent with the pathogenesis of CS. However, the presence of homologous sequences in closely related but nonpathogenic commensal spirochetes suggests that other virulence determinants may be required for pathogenesis.

Amino Acid Sequence↗