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.     

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.     

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.     

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.     

Intestinal spirochetosis: morphological characterization and cultivation of the spirochete Brachyspira aalborgi gen. nov., sp. nov.

The ultrastructure of spirochetes obtained from rectal biopsies of patients with intestinal spirochetosis was studied by means of negative staining and ultrathin sectioning. The cells were sigmoidal with tapered ends, 2 to 6 microns long, with a wavelength of 2 microns. Four flagella were inserted at each end of the cells. The maximal cell width was about 0.2 microns. The spirochetes were cultured on tryptose soy blood agar plates. They were anaerobic and grew, although very slowly, at 37 to 38.5 degrees C in an atmosphere of 5% CO2-95% H2. Two types of colonies could be distinguished. The growth characteristics and the morphology of the isolated spirochetes differ from those of previously isolated spirochetal strains. Consequently, it is proposed that the present strains constitute a new genus, Brachyspira, of the family Treponemataceae. The type species is Brachyspira aalborgi, the type strain of which is 513A (NCTC 11492).     

Comparative prevalences of Brachyspira aalborgi and Brachyspira (Serpulina) pilosicoli as etiologic agents of histologically identified intestinal spirochetosis in Australia

DNA from gastrointestinal biopsy specimens from 28 Australian patients with histologic evidence of intestinal spirochetosis (IS) was subjected to PCRs to amplify segments of the 16S rRNA and NADH oxidase genes of Brachyspira aalborgi and Brachyspira (Serpulina) pilosicoli. B. aalborgi was identified in specimens from 24 (85.7%) patients and B. pilosicoli in those from 4 (14.3%) patients (2 of whom were also positive for B. aalborgi). For two patients, no product was amplified. This study demonstrates that B. aalborgi is much more commonly involved in histologically identified IS in Australian patients than is B. pilosicoli. This is the first report of amplification of B. pilosicoli DNA from humans with IS.     

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.     

Colonic spirochetosis in children and adults

We undertook a retrospective analysis of colonic spirochetosis in 14 cases: females, 3; males, 11; children, 4; adults, 10. Two men had HIV infections. All children and both HIV-infected men had abdominal complaints, diarrhea, or both. Most other adults underwent colonoscopy for polyp screening (n = 4) or follow-up of Crohn disease (n = 1) or had other indications (n = 2) or diarrhea (n = 1). Histologically, spirochetosis was identified in all parts of the colon and was not strongly associated with active inflammation, mucosal injury, or changes of chronicity. Genotype analysis of 13 cases showed that 11 resulted from Brachyspira aalborgi and 2 from Brachyspira pilosicoli infections. Only 2 patients were treated specifically with antibiotics, with complete resolution of abdominal symptoms in 1 patient with follow-up. Follow-up biopsy result were available for 2 patients who did not receive treatment; one showed persistent spirochetosis, and the other was negative. Spirochetosis in this series had a male predominance, was generally caused by B aalborgi, and occurred in 2 distinct clinical settings: children who often have abdominal symptoms and adults who typically are asymptomatic. While treatment information remains limited, treatment can lead to resolution of symptoms in some cases.     

Bloodstream infection due to Brachyspira pilosicoli in a patient with multiorgan failure

Brachyspira pilosicoli is an etiological agent of human intestinal spirochetosis. Bloodstream infection due to this microorganism is rare. We report a case of B. pilosicoli bacteremia in a 70-year-old patient who presented with multiorgan failure.     

Identification of Brachyspira hyodysenteriae and other pathogenic Brachyspira species in chickens from laying flocks with diarrhea or reduced production or both

Cecal samples from laying chickens from 25 farms with a history of decreased egg production, diarrhea, and/or increased feed conversion ratios were examined for anaerobic intestinal spirochetes of the genus Brachyspira. Seventy-three samples positive in an immunofluorescence assay for Brachyspira species were further examined using selective anaerobic culture, followed by phenotypic analysis, species-specific PCRs (for Brachyspira hyodysenteriae, B. intermedia, and B. pilosicoli), and a Brachyspira genus-specific PCR with sequencing of the partial 16S rRNA gene products. Brachyspira cultures were obtained from all samples. Less than half of the isolates could be identified to the species level on the basis of their biochemical phenotypes, while all but four isolates (5.2%) were speciated by using PCR and sequencing of DNA extracted from the bacteria. Different Brachyspira spp. were found within a single flock and also in cultures from single chickens, emphasizing the need to obtain multiple samples when investigating outbreaks of avian intestinal spirochetosis. The most commonly detected spirochetes were the pathogenic species B. intermedia and B. pilosicoli. The presumed nonpathogenic species B. innocens, B. murdochii, and the proposed “B. pulli” also were identified. Pathogenic B. alvinipulli was present in two flocks, and this is the first confirmed report of B. alvinipulli in chickens outside the United States. Brachyspira hyodysenteriae, the agent of swine dysentery, also was identified in samples from three flocks. This is the first confirmed report of natural infection of chickens with B. hyodysenteriae. Experimental infection studies are required to assess the pathogenic potential of these B. hyodysenteriae isolates.     

Certain canine weakly beta-hemolytic intestinal spirochetes are phenotypically and genotypically related to spirochetes associated with human and porcine intestinal spirochetosis.

Four canine weakly beta-hemolytic intestinal spirochetes associated with intestinal spirochetosis (IS-associated WBHIS) were compared with IS-associated human and porcine WBHIS and the type species for Serpulina hyodysenteriae and S. innocens by using phenotypic and genotypic parameters. The IS-associated canine, human, and porcine WBHIS belonged to a phyletic group distinct from but related to previously described Serpulina type species.     

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 β-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.     

Proposed pathogenic mechanism for the diarrhea associated with human intestinal spirochetes

Spirochetes resembling Brachyspira aalborgi were found in the feces and rectal biopsies of a patient with persistent diarrhea. Although the organism failed to grow on bacteriologic media, it was found attached to the surfaces of the epithelial cells on the rectal lumen. Blunting and destruction of the cellular microvilli was evident. These induced pathologic cell surface changes, together with the presence of intracellular bacteria in the cells of the rectal colon, suggest a pathogenic mechanism for the persistent diarrhea often associated with this condition. Both the spirochetosis and clinical symptoms disappeared on treatment with metronidazole.     

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.     

Human intestinal spirochetes are distinct from Serpulina hyodysenteriae.

Twenty-nine intestinal spirochetes isolated from Australian aboriginal children and six strains from Italian adults (HRM1, -2, -4, -5, -7, and -14) were genetically examined at 15 enzyme loci by using multilocus enzyme electrophoresis. Results were compared with those previously obtained for 188 porcine intestinal spirochetes. DNA from human strain HRM7 and porcine strain Serpulina hyodysenteriae P18A were also radioactively labeled and hybridized with DNA from 12 other human and porcine intestinal spirochetes. Both the multilocus enzyme electrophoresis and hybridization techniques demonstrated that the human spirochetes were not S. hyodysenteriae. They belonged to another distinct genetic group of spirochetes that included P43/6/78, the bacterium recovered from the first recorded case of porcine intestinal spirochetosis. Bacteria in this distinct group also differed from Serpulina spp. in possessing only four, five, or occasionally six axial filaments, being slightly thinner, and having more pointed ends. These findings add further weight to the possibility that human intestinal spirochetes may act as enteric pathogens.     

Human intestinal spirochetosis

Whenever biopsy material obtained from endoscopically normal colorectal mucosa reveals the blue haematoxyphilic line between the microvilli of the covering epithelium, the rare condition of intestinal spirochetosis is diagnosed. The classification of the bacteria detected with the aid of special stains (e.g. the Warthin Starry silver stain) and in the electron microscope, continues to be something of a problem. A further point of contention is the question whether this spirochetal infection is of pathological significance or not. A point mitigating against pathogenicity is the fact that no histological signs of an inflammatory reaction are to be seen. Also, the symptoms of patients with intestinal spirochetosis are such that they provide no basis for a pronouncement on whether the infection is of a pathological or a pathological nature. On the other hand, however, a number of studies do seem to indicate that the spirochetes might be the cause of such symptoms as diarrhoea, constipation and abdominal pain. A point that would appear to support this view is the fact that such symptoms may disappear after successful treatment with metronidazole. The histological diagnosis is easily established when, faced by an apparently normal histological appearance of the colorectal mucosa, the pathologist considers the possibility of spirochetosis, and undertakes a specific search for the blue haematoxyphilic line in the covering epithelium of the colorectal mucosa.     

Rapid detection and identification of Brachyspira aalborgi from rectal biopsies and faeces of a patient

This study reports for the first time the detection of Brachyspira aalborgi in faeces and rectal biopsies of a female suffering for 3-4 months of abdominal pain with long-standing mucosal diarrhoea, rectal bleeding and suspected carcinoma of the rectum. After pre-treatment of samples (faeces and biopsies) with a liquid medium (trypticase soy broth-TSB) containing foetal calf serum (FCS, 10%) and spectinomycin and rifampicin (TSB-SR) the first detection of B. aalborgi isolate HBS1 was observed after 48 h in the primary plates of selective blood agar modified medium (BAM) containing spectinomycin and rifampicin (BAM-SR), where growth zones were signalled by a small weakly beta-haemolytic halo. Attempts to subculture spirochaetes in agar media failed. The new HBS1 isolate was only propagated in TSB broth and at electron microscopy it showed 4 endoflagella inserted at each tapered end. The phenotypic characterization of HBS1 demonstrated absence of hippurate hydrolysis, indole production, alpha-galactosidase, alpha- and beta-glucosidase activities in accordance with the B. aalborgi type strain. Rapid identification of B. aalborgi isolate HBS1 was performed directly from faeces and rectal biopsies and subsequently from pure cultures by a genetic method based on 16S DNA restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR). The sequence of 16S DNA amplicon of the isolate HBS1 was found 99.2% corresponding to that of the B. aalborgi type strain. Our results encourage further investigations for the development of a suitable selective agar medium for the isolating and cultivating B. aalborgi from human specimens.     

Intestinal spirochaetosis associated with hyperplastic and adenomatous colonic polyps

Intestinal spirochaetosis is a human colonic infection due to Brachyspira aalborgi and Brachyspira pilosicoli causing various abdominal complaints. Although the presence of healthy epithelial cells was hypothesized to be essential for the adhesion of spirochaetes to the colonic mucosa, their adhesion to hyperplastic and adenomatous colonic polyps has been observed recently. We report a case of a woman with long-standing abdominal symptoms, in whom spirochaetes were found on the colonic mucosa surrounding an adenocarcinoma in the biopsies collected during eight years of follow-up. Spirochaetes were found attached to normal mucosa, to hyperplastic and to adenomatous polyps, but not to the epithelium of the carcinoma. The rectal biopsy collected during the last follow-up colonoscopy was subjected to histopathology and to a specific examination for brachyspires, demonstrating the presence of B. pilosicoli DNA. This report could stimulate microbiological investigations during the follow-up of colonic polyps in order to explain whether the persistence of abdominal symptoms in such patients could be caused by a colonic spirochaetosis susceptible to eradication by a targeted therapy.     

Molecular and ultrastructural characterization of porcine hippurate-negative Brachyspira pilosicoli

Brachyspira pilosicoli, the causative agent of porcine intestinal spirochetosis, usually has hippurate-cleaving capacity. We have regularly isolated hippurate-negative B. pilosicoli from cases of porcine diarrhea. In this study, we show that these biochemically atypical B. pilosicoli isolates can be classified as B. pilosicoli. 16S ribosomal DNA was partially sequenced from eight hippurate-negative and two hippurate-positive B. pilosicoli-like isolates from seven herds. The differences in nucleotide sequence with B. pilosicoli P43/6/78 type strain were not associated with hippurate cleavage. In 877 bp, the hippurate-negative isolates had a similarity of 98.63 to 100% to the type strain, with the corresponding figures for the two hippurate-positive isolates being 98.86 and 100%. The nucleotide sequences of hippurate-positive isolates were identical to the respective sequences of hippurate-negative isolates from one herd. The DNA macrorestriction patterns of a total of 20 hippurate-negative and -positive B. pilosicoli isolates were diverse, and no clustering in conjunction with the hippurate reaction was found. In two herds, hippurate-positive and -negative B. pilosicoli isolates had a common macrorestriction pattern. The ultrastructure of hippurate-negative isolates was similar to the type strain. In conclusion, B. pilosicoli can be either hippurate positive or negative and, thus, the scheme for biochemical differentiation of porcine Brachyspira should be revised to include identification of hippurate-negative B. pilosicoli.