Sequence Variation of Rare Outer Membrane Protein ß-Barrel Domains in Clinical Strains Provides Insights into the Evolution of Treponema pallidum subsp. pallidum, the Syphilis Spirochete.

In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum, the syphilis spirochete, and identifying its surface-exposed ß-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of ß-barrel-encoding regions of tprC, tprD, and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 (tp0548) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) ß-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored ß-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane ß-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development.IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum, little is known about how their surface-exposed, ß-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the ß-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within ß-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.

Long-Term In Vitro Culture of the Syphilis Spirochete Treponema pallidum subsp. pallidum.

Investigation of Treponema pallidum subsp. pallidum, the spirochete that causes syphilis, has been hindered by an inability to culture the organism continuously in vitro despite more than a century of effort. In this study, long-term logarithmic multiplication of T. pallidum was attained through subculture every 6 to 7 days and periodic feeding using a modified medium (T. pallidum culture medium 2 [TpCM-2]) with a previously described microaerobic, rabbit epithelial cell coincubation system. Currently, cultures have maintained continuous growth for over 6 months with full retention of viability as measured by motility and rabbit infectivity. This system has been applied successfully to the well-studied Nichols strain of T. pallidum, as well as to two recent syphilis isolates, UW231B and UW249B. Light microscopy and cryo-electron microscopy showed that in vitro-cultured T. pallidum retains wild-type morphology. Further refinement of this long-term subculture system is expected to facilitate study of the physiological, genetic, pathological, immunologic, and antimicrobial susceptibility properties of T. pallidum subsp. pallidum and closely related pathogenic Treponema species and subspecies.IMPORTANCE Syphilis, a sexually transmitted disease with a global distribution, is caused by a spiral-shaped bacterium called Treponema pallidum subspecies pallidum Previously, T. pallidum was one of the few major bacterial pathogens that had not been cultured long-term in vitro (in a test tube), greatly hindering efforts to better understand this organism and the disease that it causes. In this article, we report the successful long-term cultivation of T. pallidum in a tissue culture system, a finding that is likely to enhance our ability to obtain new information applicable to the diagnosis, treatment, and prevention of syphilis.

The abundance of the Lyme disease pathogen Borrelia afzelii declines over time in the tick vector Ixodes ricinus.

BACKGROUND: The population dynamics of vector-borne pathogens inside the arthropod vector can have important consequences for vector-to-host transmission. Tick-borne spirochete bacteria of the Borrelia burgdorferi (sensu lato) species complex cause Lyme borreliosis in humans and spend long periods of time (>12 months) in their Ixodes tick vectors. To date, few studies have investigated the dynamics of Borrelia spirochete populations in unfed Ixodes nymphal ticks. METHODS: Larval ticks from our laboratory colony of I. ricinus were experimentally infected with B. afzelii, and killed at 1 month and 4 months after the larva-to-nymph moult. The spirochete load was also compared between engorged larval ticks and unfed nymphs (from the same cohort) and between unfed nymphs and unfed adult ticks (from the same cohort). The spirochete load of B. afzelii in each tick was estimated using qPCR. RESULTS: The mean spirochete load in the 1-month-old nymphs (~14,000 spirochetes) was seven times higher than the 4-month-old nymphs (~2000 spirochetes). Thus, the nymphal spirochete load declined by 80% over a period of 3 months. An engorged larval tick acquired ~100 spirochetes, and this population was 20 times larger in a young, unfed nymph. The spirochete load also appeared to decline in adult ticks. Comparison between wild and laboratory populations found that lab ticks were more susceptible to acquiring B. afzelii. CONCLUSION: The spirochete load of B. afzelii declines dramatically over time in domesticated I. ricinus nymphs under laboratory conditions. Future studies should investigate whether temporal declines in spirochete load occur in wild Ixodes ticks under natural conditions and whether these declines influence the tick-to-host transmission of Borrelia.

An In Vitro Blood-Feeding Method Revealed Differential Borrelia turicatae (Spirochaetales: Spirochaetaceae) Gene Expression After Spirochete Acquisition and Colonization in the Soft Tick Ornithodoros turicata (Acari: Argasidae).

In the Midwestern, Southwestern, and Southern part of the United States, the soft tick Ornithodoros turicata transmits the spirochete Borrelia turicatae, the causative agent of relapsing fever in humans. In this study, we report a simplified and an efficient method of in vitro feeding to evaluate O. turicata-B. turicatae interactions. Both nymphal and adult female ticks successfully acquired spirochetes upon in vitro feeding on the B. turicatae-infected blood. We also noted transstadial transmission of spirochetes to adult ticks that were molted from nymphs fed on B. turicatae-infected blood. A differential expression pattern for some of the B. turicatae genes was evident after acquisition and colonization of the vector. The levels of arthropod-associated lipoprotein Alp-mRNA were significantly upregulated and the mRNA levels of factor H binding protein FhbA and immunogenic protein BipA were significantly downregulated in the spirochetes after acquisition into ticks in comparison with spirochetes grown in culture medium. In addition, genes such as bta124 and bta116 were significantly upregulated in spirochetes in unfed ticks in comparison with the levels noted in spirochetes after acquisition. These findings represent an efficient in vitro blood-feeding method to study B. turicatae gene expression after acquisition and colonization in these ticks. In summary, we report that B. turicatae survive and develop in the tick host when acquired by in vitro feeding. We also report that B. turicatae genes are differentially expressed in ticks in comparison with the in vitro-grown cultures, indicating influence of tick environment on spirochete gene expression.

Development of a modified selective medium to enhance the recovery rate of Brachyspira hyodysenteriae and other porcine intestinal spirochaetes from faeces.

AIMS: The aim of this study was to develop a modified selective medium to improve the recovery rate of Brachyspira hyodysenteriae and other clinically significant intestinal spirochaetes from porcine faeces. METHODS AND RESULTS: The susceptibility of five Brachyspira spp. type strains and five Thai field isolates of B. hyodysenteriae to the antimicrobials halquinol and flavomycin was determined by in vitro susceptibility tests in the agar dilution method, and optimal incorporation rates were confirmed by broth dilution. All the spirochaetes were susceptible to halquinol at ≤ 1 µg ml(-1), while 16 µg ml(-1) of flavomycin (F) allowed their growth, and therefore, only the latter was selected for further use. F and different combinations of colistin (C), spectinomycin (S) and rifampacin (R) were incorporated into pre-enrichment broths and/or agar plates, and growth of the spirochaetes from seeded faeces was determined. Two solid media were selected for further testing using faeces from 90 finishing pigs on 10 farms. A previously recommended method of pre-enrichment did not increase the recovery rate. The use of blood agar modified medium (BAM) containing F (16 µg ml(-1)), S (400 µg ml(-1)), R (30 µg ml(-1)) and colistin (C, 100 U ml(-1)) (assigning as BAM-CSRF) reduced the growth of contaminating intestinal microbiota and resulted in a significantly higher rate of spirochaete recovery than the previous recommended medium. CONCLUSION: BAM-CSRF is a useful new selective medium for the isolation of B. hyodysenteriae and other intestinal spirochaetes from pig faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The new selective medium for isolating B. hyodysenteriae and other Brachyspira spp. from pig faeces will improve their recovery and subsequent disease diagnosis.

Novel spirochetes isolated from mosquitoes and black flies in the Czech Republic.

During the years 1999-2002, a total of 4,898 individuals of 26 species of hematophagous insects (4,149 mosquitoes, 583 black flies, and 166 tabanid flies) was examined for the presence of spirochetes using dark-field microscopy. There was an overall recovery of spirochetes from the midguts of Culicidae and Simuliidae of 23.5% and 11.4%, respectively. Spirochetes were not detected in Tabanidae. Seven spirochetal strains have been successfully recovered from mosquitoes and black flies: BR149 (Culex pipiens), BR151 (Cx. pipiens), BR173 (Cx. pipiens), BR177 (Cx. pipiens), BR193 (Aedes cinereus), BR208 (Cx. pipiens), and BR231 (Simulium noelleri). The strains have been adapted to laboratory conditions (BSK-H Complete medium). Their preliminary determination based on 16S rRNA gene sequencing has shown that they differ from the Lyme disease spirochete Borrelia burgdorferi sensu lato as well as other members of the Order Spirochaetales indicating novel bacterial species in the Family Spirochaetaceae.

The effect of fermentable carbohydrates on experimental swine dysentery and whip worm infections in pigs.

An experiment was conducted to study the effect of diets with contrasting fermentability in the large intestine on experimental infections with Brachyspira hyodysenteriae, the causative agent of swine dysentery, and the whip worm, Trichuris suis, in pigs. Two diets with organically grown ingredients were composed. Both diets were based on triticale and barley and supplemented with either rape seed cake (Diet 1) or dried chicory root and sweet lupins (Diet 2). The study had a three-factorial design, with eight groups of pigs receiving Diet 1 or Diet 2, +/-B. hyodysenteriae, and +/-T. suis. Pigs fed Diet 2 and challenged with B. hyodysenteriae did not develop swine dysentery and B. hyodysenteriae was not demonstrated in any of the pigs during the study. In contrast, 94% of the B. hyodysenteriae challenged pigs fed Diet 1 showed clinical symptoms of swine dysentery and all the pigs were shedding B. hyodysenteriae in faeces at some points in time during the experiment. The number of T. suis was lower in pigs fed Diet 2 compared to pigs fed Diet 1, but the differences were not significant. Pigs on Diet 1 and challenged with both pathogens showed clinical symptoms of SD for a longer period than pigs inoculated with B. hyodysenteriae only. The study showed that diets supplemented with highly fermentable carbohydrates from dried chicory roots and sweet lupins can protect pigs against developing swine dysentery, but do not have any significant influence on T. suis.

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.

Spherical body formation in the spirochaete Brachyspira hyodysenteriae.

When cultures of Brachyspira hyodysenteriae were grown under a wide range of in vitro conditions, at least 1% of the cells formed spherical bodies different to the normal helical form. This percentage increased considerably in aging cultures or following their incubation in caramelized media. Spherical body formation was initiated from a terminal localized swelling of the outer sheath followed by a retraction of the protoplasmic cylinder into the resulting swollen vesicle. As this occurred, the periplasmic flagella seemed to unwind from the protoplasmic cylinder. Once retracted, the protoplasmic cylinder was found to be wrapped in an organized manner around the inner surface of the membrane of the swollen vesicle. Although most were 2-3 microm in diameter, some much larger spherical bodies (6-12 microm diameter) were occasionally seen, with a corresponding increase in the visible number of peripheral protoplasmic cylinder cross-sections. Spherical bodies from older cultures did not contain protoplasmic cylinders arranged around the periphery, but instead were characterized by the presence of a centrally located, electron-dense body c. 0.5-0.8 mum in diameter. Brachyspira hyodysenteriae spherical bodies differ in both their structural organization and probable method of formation from similar structures described in other spirochaete genera.

Immunopathological investigations on bovine digital epidermitis.

Paraffin-embedded fragments of bovine digital skin lesions were sectioned and stained with Warthin-Starry, haematoxylin and eosin, Grocott's methenamine silver and immunohistochemical techniques. Microorganisms observed in the silver-stained sections were classified into four major morphological groups. Spirochaetes were the most prevalent organisms, but bacillary and coccoid elements were also present in most sections. Immunohistochemical probing demonstrated that approximately 80 per cent, 46 per cent and 41 per cent of the digital and interdigital dermatitis sections stained positively with polyclonal antisera to Treponema pallidum, Campylobacter jejuni and Fusobacterium necrophorum, respectively. An unidentified branching filamentous organism (presumed to be an actinomycete) was consistently present in the sections of samples from mild interdigital lesions.

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.

Relapsing fever-like spirochetes infecting European vector tick of Lyme disease agent.

To determine whether relapsing fever-like spirochetes associated with hard ticks may infect Ixodes ricinus ticks in central Europe, we screened questing ticks for 16S rDNA similar to that of Asian and American relapsing fever-like spirochetes. We compared the prevalence of these spirochetes to that of Lyme disease spirochetes transmitted by the same vector. Relapsing fever-like spirochetes infect 3.5% of questing vector ticks in our three central European sites near the Rhein Valley. These spirochetes differ genetically from their American and Asian analogs while being relatively homogeneous in the region we sampled. The Lyme disease genospecies most commonly detected in central Europe are distributed broadly, whereas those that are less frequently found appear to be place-specific. The absence of co-infected ticks suggests that relapsing fever-like and Lyme disease spirochetes may not share hosts. Exposure risk for relapsing fever-like spirochetes is similar to that of certain Lyme disease genospecies. Although many persons may be bitten by ticks infected by relapsing fever-like spirochetes, health implications remain unknown.

Evaluation of selective media for the isolation of Brachyspira aalborgi from human faeces.

The purposes of this study were to identify a solid medium that supports improved growth of the anaerobic intestinal spirochaete Brachyspira aalborgi, to modify this for use as a selective isolation medium and then to test the medium for its effectiveness in isolating B. aalborgi from patients' faeces. Of the media evaluated, brain heart infusion agar (BHIA) with 10 % bovine blood (BB) was the most effective base-supplement combination for growth, with colonies attaining 1.2 mm in diameter by 21 days. Incubation in an anaerobic jar (94 % H(2), 6 % CO(2)) permitted growth of larger colonies than incubation in an anaerobic chamber (80 % N(2), 10 % H(2), 10 % CO(2)). Growth was improved only slightly at 38.5 degrees C compared with 37 degrees C. Selection of B. aalborgi from artificially seeded faeces was achieved equally well on eight different solid media containing spectinomycin (400 micro g ml(-1)) alone or in combinations with polymyxin B (5 micro g ml(-1)), colistin (25 micro g ml(-1)) and rifampicin (12.5 micro g ml(-1)). By using BHIA 10 % BB with spectinomycin plus polymyxin B, B. aalborgi was isolated from one of five human faecal samples that were positive for B. aalborgi by PCR amplification. This is the first report of the isolation of B. aalborgi from human faeces.

Termite symbiotic systems: efficient bio-recycling of lignocellulose.

Termites thrive in great abundance in terrestrial ecosystems and play important roles in biorecycling of lignocellulose. Together with their microbial symbionts, they efficiently decompose lignocellulose. In so-called lower termites, a dual decomposing system, consisting of the termite's own cellulases and those of its gut protists, was elucidated at the molecular level. Higher termites degrade cellulose apparently using only their own enzymes, because of the absence of symbiotic protists. Termite gut prokaryotes efficiently support lignocellulose degradation. However, culture-independent molecular studies have revealed that the majority of these gut symbionts have not yet been cultivated, and that the gut symbiotic community shows a highly structured spatial organization. In situ localization of individual populations and their functional interactions are important to understand the nature of symbioses in the gut. In contrast to cellulose, lignin degradation does not appear to be important in the gut of wood-feeding termites. Soil-feeding termites decompose humic substances in soil at least partly, but little is known about the decomposition. Fungus-growing termites are successful in the almost complete decomposition of lignocellulose in a sophisticated cooperation with basidiomycete fungi cultivated in their nest. A detailed understanding of efficient biorecycling systems, such as that for lignocellulose, and the symbioses that provide this efficiency will benefit applied microbiology and biotechnology.

In vitro susceptibility and a new point mutation associated with tylosin-resistance in Japanese canine intestinal spirochetes.

The in vitro susceptibilities of six commonly used antimicrobial agents against 29 isolates of intestinal spirochetes isolated from dogs in Japan were examined by the agar dilution technique. In addition, the genetic basis of tylosin resistance in in vitro selected resistant mutants of two reference strains and three tylosin-susceptible field isolates obtained by three successive subcultures on blood agar containing 1 microg/ml of tylosin was investigated. Carbadox was the most active (MIC: < 0.00625) of all the antimicrobial agents. Although all the isolates were susceptible to tylosin, some were resistant to erythromycin. Tiamulin, lincomycin and dimetridazole were also very active against the isolates. All the resistant isolates did not harbor any plasmids. In vitro selected tylosin-resistant mutants of previously tylosin-susceptible isolates showed a new mutation in which their adenine at the base position equivalent to 2062 of 23S rDNA of Escherichia coli has been replaced by cytosine. These findings may both provide guidance towards the proper choice of antimicrobial agents for the treatment of canine intestinal spirochetosis, and add to the understanding of the genetic basis of tylosin resistance.

Influence of in-feed zinc bacitracin and tiamulin treatment on experimental avian intestinal spirochaetosis caused by Brachyspira intermedia.

Thirty individually caged layer hens were inoculated with Brachyspira intermedia, and 20 control birds remained unchallenged. Birds received a diet containing 100 parts/10(6) zinc bacitracin (ZnB), and were monitored for 10 weeks. B. intermedia was recovered sporadically from five of the inoculated birds, and there were no significant effects on body weight, faecal water or egg production. ZnB was presumed to be indirectly inhibiting spirochaete growth, and when removed from the diet, 18 of the 30 inoculated birds rapidly became culture positive. After 4 weeks, 10 of the 30 infected birds were treated with tiamulin at 25 mg/kg for 5 days, and 10 were returned to the diet containing ZnB. Birds receiving tiamulin became spirochaete negative and maintained their egg production, but re-infection occurred. The other 20 infected birds had a significant drop in egg production, but those receiving ZnB showed a reduced colonization by B. intermedia after 3 weeks.

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.

Intestinal spirochetosis in eight pediatric patients from Southern Sweden.

Intestinal spirochetes in humans have been recognized for more than a century, but it is still a matter of debate whether they are just commensal organisms or whether they cause colorectal disease. Most descriptions to date are of adult patients, while reports in the pediatric literature have been scarce. In a retrospective study we found eight children with intestinal spirochetosis. The findings, clinical as well as pathological, with light- and electron microscopy, are presented. In all patients, a 3 microm-thick layer of spirochetes was visualised on the luminal aspect of the epithelial cells covering the enterocytes and part of the gland openings. In five of the eight cases an inflammatory cell reaction was seen by light microscopy and in one patient a picture suggesting intracytoplasmatically located spirochetes was seen by electron microscopy. Despite partial or complete destruction of microvilli, spirochetes were still able to adhere to the enterocyte membranes. In three children there was a clear correlation between treatment and relief of symptoms. In four there was partial improvement and in one child no change in bowel-related symptoms. We believe that intestinal spirochetes may cause colorectal disease in children. Possible pathogenic mechanisms are discussed.