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.     

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.     

Prevalence,disease associations and risk factors for colonization with intestinal spirochaetes (Brachyspira spp.) in flocks of laying hens in north-eastern Italy

The present study investigated the occurrence of anaerobic intestinal spirochaetes of the genus Brachyspira in laying hen flocks in Treviso province, north-eastern Italy, with respect to prevalence, spirochaete species present, disease associations and risk factors for colonization. A total of 450 faecal samples from 45 sheds on 29 laying hen farms were cultured for intestinal spirochaetes. Nineteen sheds on 12 farms contained chickens with symptoms consistent with avian intestinal spirochaetosis, including reduced egg production, wet litter and/or pasty vents. Spirochaetes were isolated from 157 (34.8%) samples from 21 (72.4%) farms, and from 32 (71.1%) sheds. From these positive samples, 189 spirochaetal isolates were speciated using three polymerase chain reaction assays and a restriction fragment polymorphism analysis of 16S rDNA polymerase chain reaction products. Overall, 52 (27.5%) isolates were identified as pathogenic Brachyspira intermedia, 26 (13.8%) as pathogenic Brachyspira pilosicoli, 93 (49.7%) as non-pathogenic (Brachyspira innocens/Brachyspira murdochii), and 18 (9.6%) were unidentified. Faeces from 14 sheds (31%) on 10 farms (34.5%) contained B. intermedia and/or B. pilosicoli, and disease consistent with avian intestinal spirochaetosis was observed in nine of these sheds on seven farms. There was a significant association (P=0.042) between the presence of spirochaetes and using deep pits rather than conveyor belts for manure disposal. Sheds housing chickens >40 weeks of age were significantly more likely to contain spirochaetes (P=0.048) and pathogenic species (P=007) than sheds housing younger chickens. A significant association (P=0.02) was found between infection with pathogenic spirochaetes and reduced egg production.     

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.     

Antimicrobial susceptibility testing of Brachyspira intermedia and Brachyspira pilosicoli isolates from Australian chickens.

Susceptibilities of predominantly Australian isolates of the pathogenic intestinal spirochaetes Brachyspira intermedia (n = 25) and Brachyspira pilosicoli (n = 17) from chickens were tested in agar dilution against four concentrations each of the antimicrobials tiamulin, lincomycin, tylosin, metronidazole, tetracycline and ampicillin. Based on available minimum inhibitory concentration (MIC) breakpoint values for Brachyspira hyodysenteriae or other Gram-negative enteric veterinary pathogens, isolates of both species generally were susceptible to tiamulin, lincomycin, metronidazole and tetracycline. Although not classed as resistant, four isolates of B. intermedia had an elevated MIC range for tiamulin (1 to 4 mg/l), 11 isolates of B. intermedia and five of B. pilosicoli had an elevated MIC range for lincomycin (10 to 50 mg/l), one isolate of B. pilosicoli had an elevated MIC range for tetracycline (10 to 20 mg/l), and one isolate of B. intermedia and five of B. pilosicoli had an elevated MIC range for ampicillin (10 to 50 mg/l). A clear lack of susceptibility to tylosin (MIC > 4 mg/l) was seen in 11 isolates each of B. intermedia and B. pilosicoli, and to ampicillin (MIC > 32 mg/l) in two isolates of B. pilosicoli. These data suggest that some resistance to common antimicrobials exists among intestinal spirochetes obtained from laying hens and supports the need of MIC data for clinical isolates before any treatment is considered.     

Experimental infection of broiler breeder hens with the intestinal spirochaete Brachyspira ( Serpulina ) pilosicoli causes reduced egg production

The pathogenic potential of the anaerobic intestinal spirochaetes Brachyspira ( Serpulina ) pilosicoli and Brachyspira innocens was evaluated in adult chickens. Thirty 17-week-old Cobb broiler breeder hens were individually caged in three groups of 10 birds. Control birds (group A) were sham inoculated with sterile broth medium. Birds in the other two groups (groups B and C) were inoculated, respectively, with an isolate of B. innocens or of B. pilosicoli . Birds were monitored daily, and killed at 41 weeks of age. Infection had no consistent effect on body weight gain, but inoculation with B. pilosicoli resulted in a transient increase in faecal water content. B. innocens infection had no effect on egg production, but B. pilosicoli infection caused a delayed onset of laying, and a highly significant reduction in egg production over the first 11 weeks of lay. This study confirms that B. pilosicoli can cause serious egg production losses in adult chickens, while B. innocens is not obviously pathogenic.     

Development of a duplex PCR assay for detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli in pig feces

A duplex PCR (D-PCR) amplifying portions of the Brachyspira hyodysenteriae NADH oxidase gene and the B. pilosicoli 16S rRNA gene was developed and then tested on DNA extracted from 178 porcine fecal samples. The feces also underwent anaerobic culture and species-specific PCRs. Fecal extraction-D-PCR detected seven additional samples containing B. hyodysenteriae and five more containing B. pilosicoli.     

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.     

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.     

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.     

Quality-control ranges for antimicrobial susceptibility testing by broth dilution of the Brachyspira hyodysenteriae type strain (ATCC 27164T)

There are no approved standards for antimicrobial susceptibility testing of the fastidious spirochete Brachyspira hyodysenteriae. An interlaboratory study was performed to establish MIC quality control ranges for six antimicrobial agents for the type strain of B. hyodysenteriae using broth dilution. The results showed that B. hyodysenteriae B78T ATCC 27164T is a suitable quality control strain. This is a first step toward standardization of methods regarding this anaerobe.     

Antimicrobial resistance in Brachyspira pilosicoli with special reference to point mutations in the 23S rRNA gene associated with macrolide and lincosamide resistance

A point mutation in the 23S rRNA gene causes macrolide and lincosamide resistance in Brachyspira hyodysenteriae. The possible occurrence of a similar mutation in Brachyspira pilosicoli was studied and the MICs of six antimicrobial agents for Swedish field isolates of B. pilosicoli were determined. Of 10 isolates with high MICs of macrolide and lincosamide antibiotics, six had a mutation in nucleotide position 2058 or 2059 in the 23S rRNA gene as compared to the wild type of Escherichia coli, whereas none of 10 tylosin-susceptible isolates were mutated in this region. The mutations found in position 2058 were A --> T transversions, and in position 2059 either A --> G transitions or A --> C transversions. The MICs at which 90% of the B. pilosicoli field isolates were inhibited by tylosin, erythromycin, clindamycin, virginiamycin, tiamulin, and carbadox, were >256, >256, >4, 4, 2, and 0.125 microg/ml, respectively. In conclusion, point mutations in positions 2058 and 2059 of the 23S rRNA gene can cause macrolide and lincosamide resistance in B. pilosicoli. Macrolide resistance is widespread among Swedish field isolates of B. pilosicoli. Notably also a few isolates with elevated MICs of tiamulin were found.     

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.     

Human intestinal spirochetosis in Japan; its incidence, clinicopathologic features, and genotypic identification.

Human intestinal spirochetosis is a common condition in Western countries, but is not well recognized in Japan. To demonstrate the incidence and clinicopathologic findings of human intestinal spirochetosis in Japan, we retrospectively investigated biopsy, and endoscopically or surgically resected specimens of the large intestine. Among a series of 2556 samples, 11 cases of human intestinal spirochetosis were detected (0.4%). Together with additional nine cases sporadically found, 20 cases of human intestinal spirochetosis were subjected to molecular detection of two strains of spirochetes (Brachyspira aalborgi and Brachyspira pilosicoli) by amplifying species-specific portion of 16S ribosomal RNA and NADH oxydase gene by polymerase chain reaction. B. aalborgi was detected in all cases examined, three of which revealed dual infection of both species. Our results suggest that human intestinal spirochetosis infection is relatively rare, and B. aalborgi is the most prevalent species in Japan. Most of human intestinal spirochetosis were asymptomatic, although symptomatic in exceptional cases. In addition, we emphasize a usefulness of immunostaining with anti-Treponema pallidum and anti-Mycobacterium bovis polyclonal antibodies for detecting the spirochetes.     

Typhlocolitis associated with spirochaetes in duck flocks

The aetiology of increased mortality observed in two breeder duck flocks (Flock A consisting of 3500 laying ducks and Flock B comprising 4300 laying ducks) during the first egg-laying season was studied. In Flocks A and B, 773 ducks and 715 ducks (18.4% and 16.6%) died within a 24-week and a 20-week period, respectively. Death was preceded by clinical signs including movement difficulties, lack of appetite and depression lasting for 1 to 2 days. Diarrhoea was not observed. On gross pathological examination, the ducks were found to have haemorrhagic to fibrinonecrotic typhlocolitis, renal degeneration accompanied by fibrosis and mineralization, hepatic and splenic amyloidosis, and swelling of some of the metatarsal and phalangeal joints. Histopathological and immunohistochemical examination consistently demonstrated spirochaetes in the mucous membrane of the affected large intestine. On the basis of their cultural and biochemical properties and polymerase chain reaction sequencing analysis, four out of seven spirochaete strains isolated from the ducks (Flock A) by culture on special media under anaerobic conditions were identified as Brachyspira hyodysenteriae, and five out of eight strains (Flock B) were identified as Brachyspira pilosicoli. This is the first report on the isolation of B. hyodysenteriae and B. pilosicoli from laying ducks affected by fibrinonecrotic typhlocolitis.     

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.     

Typhlocolitis associated with spirochaetes in goose flocks.

The role of Brachyspira bacteria in the aetiology of increased mortality observed in two breeder goose flocks (Flock A consisting of 1,500 and Flock B comprising 4,500 laying geese) at the end of the first egg-laying season, in the period of moulting, was studied. In Flock A 415 geese (28%) died during an 8-week period while in Flock B 834 geese (18%) died during a 12-week period. On gross pathological examination, the geese were found to have haemorrhagic-to-necrotic inflammation of the large intestine (colon and rectum) and fibrinonecrotic typhlitis accompanied by severe degeneration. Often, fibrosis of the kidneys, and in five of the geese secondary visceral urate deposition ("visceral gout") was also observed. Histopathological examination consistently demonstrated spirochaetes in the mucous membrane of the affected large intestine. This was confirmed by the results of immunohistochemical and electron microscopic examination. In addition, Trichomonas stages were also detected from the large intestine of 11 geese. On the basis of their cultural and biochemical properties, and PCR sequencing analysis, eight out of the nine spirochaete strains isolated from the geese by culture on special media under anaerobic conditions were identified as Brachyspira alvinipulli. This is the first report on the isolation of B. alvinipulli from laying geese affected with fibrinonecrotic typhlocolitis.     

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.     

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.