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PubMed1995

Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae.

Serpulina (Treponema) hyodysenteriae is the causative agent of swine dysentery, a contagious mucohemorrhagic disease of the colon. Diagnosis of swine dysentery is extremely difficult because of the presence of cross-reactive antibodies to the proteins of S. hyodysenteriae and Serpulina innocens, a nonpathogenic inhabitant of the porcine large intestine. Therefore, monoclonal antibodies (MAbs) against the serotype-specific lipooligosaccharide (LOS) antigens of S. hyodysenteriae were produced to rapidly differentiate S. hyodysenteriae from S. innocens. Whole-cell preparations of S. hyodysenteriae serotypes 1 through 7 were used as antigens. MAbs were characterized by an indirect enzyme-linked immunosorbent assay with whole-cell or LOS antigen and by Western blot (immunoblot) analysis with whole-cell lysates as antigen. A total of 12 LOS-specific MAbs which could identify and differentiate the seven original serotypes of S. hyodysenteriae were produced. The MAb serospecificities are as follows: MAb 9G8, serotype 1; MAb 31D9, serotype 2; MAb 7D3, serotypes 2 and 7; MAb 24B7, serotype 3; MAb 13C2, serotype 4; MAb 18E9, serotype 4; MAb 2B7, serotype 6; MAb 1D2, serotypes 2, 5, and 7; MAb 9C5, serotypes 2, 5, and 7; MAb 11C9, serotype 7; MAb 11E10, serotype 7; and MAb 6G11, serotype 7.

Animals↗
PubMed1996

Enhanced isolation of Serpulina hyodysenteriae by using sliced agar media.

A method has been developed for separating Serpulina hyodysenteriae, a large spirochete and the causative agent of swine dysentery (SD), from other fecal anaerobic bacteria in rectal and colonic swabs. This was done by cutting the blood agar in parallel cuts and streaking perpendicular to the cuts in the center of the petri dish. Migration of S. hyodysenteriae from the central streak was apparent by the presence of strong beta-hemolysis along the edges of the cuts. If only S. hyodysenteriae migrated in the cut, they migrated to the end of the cut. However, if both motile bacteria and S. hyodysenteriae migrated in the cut, the motile bacteria migrated to the end of the cut where they formed colonies and the S. hyodysenteriae located along the edges of the cut between the colonies of motile bacteria and the central streak. Although motile bacteria were present where S. hyodysenteriae located, the growth of the motile bacteria was partially inhibited since they rarely formed visible colonies and were low in number. The cut in the agar was thought to improve traction for the serpentine movement of the S. hyodysenteriae and for the flagellar movement of the motile bacteria. Use of sliced blood agar was superior to conventionally streaked blood agar in that (i) it was easier to see strong beta-hemolysis on sliced agar; (ii) frequently, a confirmatory diagnosis could be made using only one petri dish with sliced agar, thereby saving time and media; (iii) S. hyodysenteriae could sometimes be isolated free of other bacteria; and (iv) sliced agar was more effective in isolating S. hyodysenteriae from swine with chronic diarrhea and nondiarrhetic carriers of SD in which the shedding of S. hyodysenteriae was low.

Agar↗
PubMed1997

Genetic and phenotypic characterization of intestinal spirochetes colonizing chickens and allocation of known pathogenic isolates to three distinct genetic groups.

Infection with intestinal spirochetes has recently been recognized as a cause of lost production in the poultry industry. Little is known about these organisms, so a collection of 56 isolates originating from chickens in commercial flocks in Australia, the United States, The Netherlands, and the United Kingdom was examined. Strength of beta-hemolysis on blood agar, indole production, API ZYM enzyme profiles, and cellular morphology were determined, and multilocus enzyme electrophoresis was used to analyze the extent of genetic diversity among the isolates. The results were compared with those previously obtained for well-characterized porcine intestinal spirochetes. The chicken isolates were genetically heterogeneous. They were divided into 40 electrophoretic types distributed among six diverse genetic groups (groups b to g), with a mean genetic diversity of 0.587. Strains in two groups (groups d and e) may represent new species of Serpulina, and the groups contained only strains isolated from chickens. Three genetic groups contained isolates previously shown to be pathogenic for chickens. These corresponded to the proposed species "Serpulina intermedius," to an unnamed group (group e), and to Serpulina pilosicoli. Two of the chicken isolates (one "S. intermedius" and one S. pilosicoli isolate) were strongly beta-hemolytic, two (both "S. intermedius") had an intermediate level of beta-hemolysis, and the rest were weakly beta-hemolytic. Fourteen isolates of "S. intermedius" produced indole, as did one isolate from group d. Isolates identified as S. pilosicoli resembled porcine isolates of this species, having four to six periplasmic flagella inserted subterminally in a single row at each end of the cell, and had tapered cell ends. All other spirochetes were morphologically similar, having seven or more periplasmic flagella and blunt cell ends. The identification of three genetic groups containing pathogenic isolates provides an opportunity for more detailed epidemiologic studies with these pathogens and for the development of improved diagnostic tests.

Animals↗
PubMed1997

Identification of Serpulina species associated with porcine colitis by biochemical analysis and PCR.

A PCR system for the detection and identification of group IV spirochetes (Serpulina pilosicoli) was designed to complement biochemical tests, e.g., the hippurate hydrolysis and beta-glucosidase tests, and to verify the accuracy of a previously proposed biochemical classification system. The PCR assay was based on amplification of a segment of the 16S rRNA gene. Both primers were constructed to selectively amplify the 16S rRNA gene of Serpulina pilosicoli. All analyzed Serpulina strains exhibiting the capacity to hydrolyze hippurate and lacking beta-glucosidase activity, including the type strain for spirochetal diarrhea, P43, were amplified with the PCR system. All other tested strains, including type and field strains of different phenotypes of Serpulina species, as well as Salmonella species, Campylobacter species, and Escherichia coli strains, were negative in the assay. Among the tested strains were 18 Scottish field isolates originating from the mucosae of pigs with colitis. A simple classification scheme, suitable for routine classification of porcine intestinal spirochetes, is also presented. The scheme is based on hemolysis, indole production, and the hippurate hydrolysis test.

Animals↗
PubMed1997

Identification and characterization of Serpulina pilosicoli isolates recovered from the blood of critically ill patients.

The phenotypic and genetic characteristics of spirochetes isolated from the blood of one U.S. and six French patients with severe clinical disease or impaired immunity were examined. All spirochetes were anaerobic, weakly beta-hemolytic, positive for hippurate hydrolysis, and negative for beta-glucosidase activity. Cell lengths ranged from 4 to 8 microm, and each isolate had between 8 and 12 periplasmic flagella per cell. These features were consistent with the spirochetes' being Serpulina pilosicoli, the agent of intestinal spirochetosis. All isolates were positive in a PCR assay amplifying a portion of the S. pilosicoli 16S rRNA gene, and they all grouped with fecal isolates of S. pilosicoli in multilocus enzyme electrophoresis (MLEE). The blood isolates could be differentiated from each other by MLEE, although the U.S. and two French isolates were closely related. Apparently S. pilosicoli may translocate from the large intestine to establish spirochetemia. The clinical significance of this finding remains uncertain and requires further investigation.

Bacteremia↗
PubMed1998

Isolation of Serpulina pilosicoli from rectal biopsy specimens showing evidence of intestinal spirochetosis.

Histologic evidence of intestinal spirochetosis (IS) was found in 22 of 41 (53.7%) rectal biopsy specimens from homosexual men attending a sexually transmitted diseases clinic. Serpulina pilosicoli was cultured from 11 of the IS-positive biopsy specimens (50%) and from 2 specimens (10.5%) in which spirochetes were not observed. The association between seeing spirochetes in biopsy specimens and isolating S. pilosicoli was statistically significant, clearly indicating that this spirochete is the agent of IS.

Biopsy↗
PubMed1998

Typhlitis caused by intestinal Serpulina-like bacteria in domestic guinea pigs (Cavia porcellus).

Between January 1992 and December 1996, Serpulina-like bacteria were demonstrated in intestinal tract lesions from 37 of 88 guinea pigs submitted to the University of Ghent in Ghent, Belgium, for necropsy because of disease and death from different unknown causes. All infected animals had a history of sudden death with minimal introductory clinical signs. Occasionally, they produced yellow, slimy feces or showed nervous signs, but the condition always had a fatal outcome within 24 h. When larger colonies of guinea pigs were involved, the disease spread very rapidly unless treatment with ronidazole was initiated. Lesions consisted of a catarrhal or hemorrhagic inflammation of the colon and cecum (typhlitis). Electron microscopy demonstrated the presence of large numbers of Serpulina-like organisms adhering to the cecal mucosae of these animals. Attempts to isolate the agents failed. The organisms did not stain by an immunofluorescence technique for the detection of Serpulina hyodysenteriae. The present data provide evidence that intestinal Serpulina-like organisms can be important as a cause of disease in guinea pigs.

Animals↗
PubMed1998

Canine intestinal spirochetes consist of Serpulina pilosicoli and a newly identified group provisionally designated "Serpulina canis" sp. nov.

The spirochetes inhabiting the large intestines of humans and animals consist of a diverse group of related organisms. Intestinal spirochetosis caused by Serpulina pilosicoli is a newly recognized enteric disease of human beings and animals with potential public health significance. The purpose of this study was to determine the species identity of canine intestinal spirochetes by comparing 30 isolates obtained from dogs in Australia (n = 25) and the United States (n = 5) with reference strains representing Serpulina species and Brachyspira aalborgi, by phenotypic and genetically based typing methods. All of the canine isolates were indole negative and produced a weak beta-hemolysis when cultured anaerobically on agar medium containing blood. Four isolates were identified as S. pilosicoli by 16S rRNA-specific PCR assays, rRNA gene restriction fragment length polymorphism or ribotyping, and multilocus enzyme electrophoresis. The remaining 26 isolates formed a cluster related to porcine Serpulina innocens as determined by multilocus enzyme electrophoresis but had a unique ribotype pattern. The data suggested the existence of a novel Serpulina species, provisionally designated "Serpulina canis," colonizing the intestines of dogs.

Animals↗
PubMed1999

PCR amplification from fixed tissue indicates frequent involvement of Brachyspira aalborgi in human intestinal spirochetosis.

PCR procedures amplifying portions of the 16S rRNA and NADH oxidase genes of Brachyspira aalborgi and Serpulina pilosicoli were applied to DNA extracted from paraffin-embedded human colonic or rectal tissues from 30 Norwegian, Australian, and U.S. patients, 16 of whom had histologic evidence of intestinal spirochetosis (IS). B. aalborgi-specific sequences were identified by PCR in 10 of the IS patients (62.5%) but none of the others, while S. pilosicoli sequences were not detected in tissues from any patient. Direct sequencing of products from three of the positive samples provided further confirmation of the presence of B. aalborgi. B. aalborgi may be a more common cause of intestinal spirochetosis than has been previously thought.

Adolescent↗
PubMed2000

Brachyspira aalborgi infection diagnosed by culture and 16S ribosomal DNA sequencing using human colonic biopsy specimens.

In this study we report on the isolation and characterization of the intestinal spirochete Brachyspira aalborgi using human mucosal biopsy specimens taken from the colon of a young adult male with intestinal spirochetosis. A selective medium, containing 400 microg of spectinomycin/ml and 5 microg of polymyxin/ml was used for the isolation procedure. A high degree of similarity, in terms of phenotypic properties and 16S ribosomal DNA sequence, was observed between the isolated strain, named W1, and the type strain, 513A, of B. aalborgi. A similarity of 99.7% in the nucleotide sequence was found between W1 and 513A(T), based on the almost-complete gene. A short segment of the 16S rRNA gene was amplified by PCR using genetic material enriched from paraffin-embedded biopsy specimens, which were taken from the patient on two occasions. The products showed 16S rRNA gene sequences virtually identical to that of strain 513A(T) in the actual region. Immunohistochemistry was performed on the colonic biopsy specimens with a polyclonal antibody raised against an intestinal spirochete isolated in a previous case of human intestinal spirochetosis. The antibody reacted strongly with the spirochete on the luminal epithelium. No immune reaction was seen within or below the surface epithelium. Routine histology did not reveal signs of colitis. Electron microscopy showed spirochetes attached end-on to the colonic mucosal surface. The isolate grew poorly on a commonly used selective medium for intestinal spirochetes, which may explain previous failures to isolate B. aalborgi.

Adult↗
PubMed2001

Diagnostic examination of human intestinal spirochetosis by fluorescent in situ hybridization for Brachyspira aalborgi, Brachyspira pilosicoli, and other species of the genus Brachyspira (Serpulina).

Human intestinal spirochetosis, characterized by end-on attachment of densely packed spirochetes to the epithelial surface of the large intestines as a fringe has been associated with the weakly beta-hemolytic spirochetes Brachyspira aalborgi and Brachyspira (Serpulina) pilosicoli. In this study, fluorescent in situ hybridization with oligonucleotide probes targeting 16S or 23S rRNA of B. aalborgi, B. pilosicoli, and the genus Brachyspira was applied to 40 sections of formalin-fixed, paraffin-embedded intestinal biopsy specimens from 23 Danish and 15 Norwegian patients with histologic evidence of intestinal spirochetosis. Five biopsy specimens from patients without intestinal spirochetosis and three samples from pigs with experimental B. pilosicoli colitis were examined as well. In addition, the 16S ribosomal DNAs of two clinical isolates of B. aalborgi were sequenced, and a PCR procedure was developed for the identification of B. aalborgi in cultures. The genotypic characteristics of the two clinical isolates showed very high (99.5%) similarity with two existing isolates, the type strain of B. aalborgi and a Swedish isolate. Hybridization with the Brachyspira genus-specific probe revealed a brightly fluorescing fringe of spirochetes on the epithelia of 39 biopsy specimens, whereas 1 biopsy specimen was hybridization negative. The spirochetes in biopsy specimens from 13 Danish and 8 Norwegian patients (55.3%) were identified as B. aalborgi. The spirochetes in the biopsy specimens from the other 17 patients hybridized only with the Brachyspira probe, possibly demonstrating the involvement of as-yet-uncharacterized Brachyspira spirochetes in human intestinal spirochetosis.

Adolescent↗
PubMed2003

Detection by PCR and isolation assays of the anaerobic intestinal spirochete Brachyspira aalborgi from the feces of captive nonhuman primates.

The purpose of this study was to investigate the presence of the anaerobic intestinal spirochetes Brachyspira aalborgi and Brachyspira pilosicoli in the feces of captive nonhuman primates (n = 35) from 19 species housed at the Zoological Gardens, Perth, Western Australia. Both spirochete species are known to infect human beings. DNA was extracted from freshly collected feces with a commercially available QIAamp DNA stool minikit and subjected to PCR protocols amplifying portions of the 16S rRNA genes of the two spirochete species. The feces were also subjected to selective culture for the spirochetes. Subsequently, feces from 62 other captive animals or birds representing 39 species at the zoo were examined by PCR to determine whether they were reservoirs of infection. Six fecal samples from individuals from four primate species (two vervet monkeys, two Tonkean macaques, one Japanese macaque, and one hamadryas baboon) tested positive in the B. aalborgi PCR. B. aalborgi was not detected by PCR in any of the other animal or bird species tested, and B. pilosicoli was not detected in the primates or any of the other animals or birds. B. aalborgi was isolated from both PCR-positive vervet monkeys. This is the first time that B. aalborgi has been isolated from nonhuman primates and the first time that it has been isolated from the feces of any species.

Animals↗
PubMed2003

Antimicrobial susceptibility testing of porcine Brachyspira (Serpulina) species isolates.

No standardized method for susceptibility testing of Brachyspira spp. is currently available. A broth dilution procedure was evaluated and used to test the activities of six antimicrobial agents for 108 isolates of Swedish porcine Brachyspira spp. representing biochemical groups I, II, and III. Group I corresponds to Brachyspira hyodysenteriae, group II corresponds to B. intermedia, and group III corresponds to B. murdochii and B. innocens. A panel was designed with the antimicrobial agents dried in tissue culture trays with wells that allowed a liquid volume of 0.5 ml in each and agitation of the broth when incubated on a shaker. The MICs were determined by using brain heart infusion broth with 10% fetal calf serum. For 10 isolates, the results obtained in broth were compared to the MICs obtained on two different types of agar. Different inoculum densities and incubation times were also compared. The concentrations at which 90% of the B. hyodysenteriae isolates (n = 72) were inhibited in the broth dilution test by tiamulin (0.25 micro g/ml), tylosin (>256 micro g/ml), erythromycin (>256 micro g/ml), clindamycin (>4 micro g/ml), virginiamycin (4 micro g/ml), and carbadox (0.06 micro g/ml) were determined. The MICs tended to be lower in broth than on agar. Differences in inoculum densities and incubation times had little influence on the MICs. The evaluated broth dilution test was simple to perform, the end points were easily read, and the results were reproducible and reliable. No isolates with decreased susceptibility to tiamulin were found among the Swedish isolates tested.

Animals↗
PubMed1992

Clinically silent infections in patients with oligoarthritis: results of a prospective study.

Oligoarticular synovitis of undetermined origin can closely resemble an incomplete form of reactive arthritis/Reiter's syndrome. Eighty three patients with oligoarthritis of undetermined origin were studied prospectively to identify asymptomatic infections potentially triggering the inflammatory response in the synovial fluid. At the time of initial evaluation, 57 (69%) of the patients with oligoarthritis and 4/20 (20%) of the control subjects were carriers of clinically silent infections. Evidence for persistent or prior chlamydial infections was frequently and exclusively found in the study group (30/83 (36%) patients v no controls), whereas undetected urogenital infections with mycoplasma were present in nine (11%) patients and four (20%) controls. Eleven (13%) of the patients carried cellular and humoral responses to Borrelia burgdorferi. The HLA-B27 haplotype represented a major risk factor for the development of oligoarthritis but not for development of sacroiliitis. Re-evaluation after one year showed that the course and outcome of the oligoarticular disease did not correlate with a specific infectious organism and were not affected by antibiotic treatment sufficient to treat the carrier state.

Acute Disease↗