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.     

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.     

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.     

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.     

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.     

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.     

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).     

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.     

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.     

Genetic similarity of intestinal spirochetes from humans and various animal species.

The chromosomal DNA of spirochetes isolated from human, swine, dog, mouse, rat, and chicken intestine or feces was subjected to restriction enzyme analysis and hybridization with three different DNA probes, derived from a flagellin gene, a hemolysin gene, and the 16S rDNA sequence of the pathogenic swine intestinal spirochete Serpulina hyodysenteriae. This genetic analysis showed that intestinal spirochetes represent a heterogeneous but related population of bacteria. In general, unique genotypes were distinguished among isolates from the same host species; they were not present among isolates from other host species. This suggests the host specificity of some strains. An exception to this are isolates from humans and dogs suffering from gastrointestinal disorders; these isolates showed highly similar or even identical genotypes. None of them resembled any of the genotypes of isolates found in other host species without apparent disease.     

Evaluation of tiamulin and lincomycin for the treatment of broiler breeders experimentally infected with the intestinal spirochaete Brachyspira pilosicoli

Brachyspira pilosicoli strain CPSp1 isolated from a chicken in a broiler breeder flock in Queensland was used to experimentally infect 30 individually caged 22-week-old Cobb 500 broiler breeder hens. Another 10 birds were sham-inoculated with sterile broth. All birds failed to become colonized. At 29 weeks of age, all birds were transferred to a diet containing 50 parts/10 6 zinc bacitracin (ZnB) and were re-challenged with the same B. pilosicoli strain at 32 weeks of age, weekly for 5 weeks. The majority of the inoculated birds then became colonized, confirming previous findings that ZnB can increase susceptibility to colonization with B. pilosicoli. The control group remained uninfected. Infected groups tended to have an increased faecal water content and faecal staining of eggshells. Ten birds were then treated by crop tube with 25 mg/kg body weight tiamulin for 5 days, and 10 birds with 20 mg/kg body weight lincomycin for 5 days. Both treatments removed the infection, while untreated birds remained infected. The results support previous observations that ZnB at 50 parts/10 6 in the diet increases the susceptibility of birds to B. pilosicoli infection, and demonstrated the usefulness of both tiamulin and lincomycin for treatment of infection with B. pilosicoli in adult birds.     

Zinc bacitracin enhances colonization by the intestinal spirochaete Brachyspira pilosicoli in experimentally infected layer hens

Brachyspira pilosicoli strain CPSp1 isolated from a chicken in a broiler breeder flock in Queensland was used to experimentally infect 40 individually caged 22-week-old laying hens. Another 10 birds were sham-inoculated with sterile broth. All chickens received a commercial layer diet, but 10 infected birds had 50 parts/10 6 zinc bacitracin (ZnB) incorporated in their food. Birds were kept for 7 weeks, and faecal moisture, egg numbers, egg weights and body weights were recorded weekly. B. pilosicoli was isolated from the faeces of only three of the 30 inoculated birds receiving the diet without ZnB, whereas seven of the 10 inoculated birds receiving ZnB in their diet were colonized. This difference in colonization rate was highly significant ( P = < 0.001). Dietary ZnB at 50 parts/10 6 therefore predisposed to colonization by B. pilosicoli. Despite colonization, no significant production differences were found between the birds in the three groups.     

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.     

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.     

Localization of Borrelia burgdorferi in the nervous system and other organs in a nonhuman primate model of lyme disease

Lyme borreliosis is caused by infection with the spirochete Borrelia burgdorferi. Nonhuman primates inoculated with the N40 strain of B. burgdorferi develop infection of multiple tissues, including the central (CNS) and peripheral nervous system. In immunocompetent nonhuman primates, spirochetes are present in low numbers in tissues. For this reason, it has been difficult to study their localization and changes in expression of surface proteins. To further investigate this, we inoculated four immunosuppressed adult Macaca mulatta with 1 million spirochetes of the N40 strain of B. burgdorferi, and compared them with three infected immunocompetent animals and two uninfected controls. The brain, spinal cord, peripheral nerves, skeletal muscle, heart, and bladder were obtained at necropsy 4 months later. The spirochetal tissue load was first studied by polymerase chain reaction (PCR)-ELISA of the outer surface protein A (ospA) gene. Immunohistochemistry was used to study the localization and numbers of spirochetes in tissues and the expression of spirochetal proteins and to characterize the inflammatory response. Hematoxylin and eosin and trichrome stains were used to study inflammation and tissue injury. The results showed that the number of spirochetes was significantly higher in immunosuppressed animals. B. burgdorferi in the CNS localized to the leptomeninges, nerve roots, and dorsal root ganglia, but not to the parenchyma. Outside of the CNS, B. burgdorferi localized to endoneurium and to connective tissues of peripheral nerves, skeletal muscle, heart, aorta, and bladder. Although ospA, ospB, ospC, and flagellin were present at the time of inoculation, only flagellin was expressed by spirochetes in tissues 4 months later. Significant inflammation occurred only in the heart, and only immunosuppressed animals had cardiac fiber degeneration and necrosis. Plasma cells were abundant in inflammatory foci of steroid-treated animals. We concluded that B. burgdorferi has a tropism for the meninges in the CNS and for connective tissues elsewhere in the body.     

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.     

安徽黄山地区中华硬蜱及其宿主感染莱姆病Borrelia afzelii的检测

利用PCR和病原体分离技术对安徽黄山地区中华硬蜱及其宿主感染莱姆病螺旋体的状况进行了检测,结果从40只黄山林区的中华硬蜱中获得了一株莱姆病螺旋体分离株,寄主动物及其他中华硬蜱均未获得阳性分离结果.以16s rRNA为靶标进行PCR检测,6.78%雌性和5%雄性中华硬蜱发现有阳性感染,2只草兔(92只)和1只麂子(16只)也发现有阳性感染;若蜱和其余宿主动物未发现阳性感染.阳性分离株和扩增获得的168 rRNA序列结果一致,同GenBank中的B.afzelii的系统发育关系最近,提示该分离株螺旋体属于B.afzelii.该结果与前期的实验传播结果提示中华硬蜱是我国南方莱姆病的传播媒介.     

Infectious typhlitis in chickens caused by spirochetes

A weakly haemolytic spirochete was detected with an unabsorbed fluorescent antiserum to Treponema hyodysenteriae in smears and cultures of scrapings of caecal mucosa of laying hens with diarrhoea. Two groups of experimental chickens were fed a pure culture of this spirochete or homogenated intestinal contents of affected birds. Both groups showed clinical signs of disease such as increased water content of faecal material and slight retardation of growth. A non-specific typhlitis which histologically resembled milder forms of swine dysentery was seen in the birds from which spirochetes were isolated. The isolate obtained differed in cultural, biochemical, anti-genic and morphological characteristics from T. hyodysenteriae. The pathological significance of intestinal spirochetes and their possible epidemiological relation to swine dysentery are discussed.