A monoclonal antibody reacting with the cell envelope of spirochaetes isolated from cases of intestinal spirochaetosis in pigs and humans

Abstract A monoclonal antibody (mAb) designed BJL/AC1 was prepared against the cell envelope of an intestinal spirochaete (strain 3295) that was isolated from a pig with intestinal spirochaetosis. The mAb reacted with a band of approximately 29 kDa in cell envelope preparations from 13 porcine and 11 human spirochaetes isolated from cases of intestinal spirochaetosis, but did not react with preparations made from a range of other intestinal spirochaetes. Immunogold labelling demonstrated that the reactive epitope was located on the cell envelope of the strains causing intestinal spirochaetosis. The mAb was used in an indirect immunofluorescence test to detect spirochaetes in the faeces of pigs with experimentally induced intestinal spirochaetosis. The mAb should prove to be a useful reagent for detection and identification of spirochaetes that are specifically associated with intestinal spirochaetosis.     

Isolation and characterization of a mercury-resistant broad-host-range plasmid from Pseudomonas cepacia

Abstract The sequence of 1383 nucleotides of the DNA encoding 16S rDNA was determined for strains of human intestinal spirochaetes, comprising an unnamed isolate and " Brachyspira aalborgi " NCTC 11492. A phylogenetic tree was inferred from aligned sequence comparisons between the intestinal spirochaetes, representatives of the Spirochaetales and Escherichia coli . The type strain of Brachyspira aalborgi , though related to the Serpulina spp. at approx. 96.5% sequence similarity was distinct and separated from the unnamed human intestinal isolate, HIS Oman, N26. The latter formed a separated and novel lineage that bisected the Spirochaetales.     

Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons

Abstract Multilocus enzyme electrophoresis (MEE) analysis and comparisons of nearly complete 16S rRNA gene sequences (1416 nucleotide positions) were used to evaluate phylogenetic relationships among Serpulina hyodysenteriae strain B78^T, S. innocens strain B256^T, Brachyspira aalborgi strain 513A^T, and eight uncharacterised strains of swine, avian, and human intestinal spirochaetes. From MEE analysis, nine strains could be assigned to five groups containing other intestinal spirochaetes ( genetic distances between groups = 0.6–0.9). Chicken spirochaete strain C1 and B. aalborgi 513A^T represented unique electrophoretic types and formed their own MEE groups. Despite MEE differences, the 11 strains had highly similar (96.3–99.9%) 16S rRNA sequences. These findings point out limitations of both MEE analysis and 16S rRNA sequence comparisons when used as solitary techniques for classifying intestinal spirochaetes related to Brachyspira/ Serpulina species.     

Antibodies to a common outer envelope antigen of Treponema hyodysenteriae with antibacterial activity

Outer envelopes of Treponema hyodysenteriae strains P18A and VS1 were prepared and characterized by SDS-PAGE. In Western blot analysis of eleven strains of T. hyodysenteriae and two intestinal non-pathogenic spirochaetes, polyclonal antiserum raised to the outer envelopes of strain P18A contained antibodies primarily to two polypeptides. A 45 kDa polypeptide was present in only two strains of T. hyodysenteriae, P18A and MC52/80, whereas another antigen of 16 kDa was common to all eleven strains of T. hyodysenteriae but was not present in the two nonpathogens. Immunogold labelling of whole organisms suggested that the 16 kDa antigen was present on the surface of the spirochaetes. In in vitro tests the serum agglutinated and inhibited growth of only the T. hyodysenteriae strains, suggesting that antibodies to the 16 kDa antigen were responsible for these activities. Serum from a gnotobiotic pig infected with T. hyodysenteriae strain P18A had antibodies to the 16 kDa antigen alone and also possessed agglutinating and growth-inhibitory activities.     

Weakly beta-haemolytic human intestinal spirochaetes antagonize the haemolytic activity of Clostridium perfringens alpha-toxin producer

The production of haemolytic antagonism between weakly beta-haemolytic human intestinal spirochaetes (wbetaHIS) related to human intestinal spirochaetosis and Clostridium perfringens alpha-toxin producer was investigated. A reduction of the clostridial haemolytic activity and a distortion of the haemolytic halo of clostridial alpha-toxin surrounded by a small zone of poorly cooperative haemolysis was clearly observed on the level of the spirochaetal growth area when 40 out of 41 wbetaHIS were cultivated in sheep blood agar plates together with Clostridium perfringens alpha-toxin producer. This phenomenon of haemolytic antagonism was observed only when wbetaHIS grew 72-96 hours sooner than C. perfringens and after the inoculum of the latter at a distance of 0 to 10 mm from wbetaHIS the plates were anaerobically incubated for an additional 48 hours and the bacteria were used at concentrations ranging from 10(7) to 10(4) CFU/ml. These results were also observed between C. perfringens and weakly beta-haemolytic intestinal spirochaetes related to animal intestinal spirochaetosis including avian strains and Brachyspira (Serpulina) pilosicoli of porcine origin.     

Comparison of peptidase, glycosidase and esterase activities of oral and non-oral Treponema species

The enzyme profiles of 20 oral and non-oral Treponema strains were investigated using an API ZYM Complete Research kit. The test included 10 2-naphthyl derivatives of fatty acids, 20 p-nitrophenol derivatives of carbohydrates and 60 2-naphthylamide derivatives of amino acids and peptides. The oral Treponema species investigated were T. denticola, T. vincentii and T. Pectinovorum. The non-oral species examined were T. phagedenis, T. hyodysenteriae and intestinal spirochaetes of human and chicken origin. Esterase activities on C5 to C10 fatty acids were common among different Treponema species. Glycosidase activities were infrequently observed in T. vincentii, T. pectinovorum and T. phagedenis Reiter strain. Arabinosidase, lactosidase and xylosidase activity was observed in the T. hyodysenteriae strains but alpha-L-fucosidase activity was found only in T. denticola and T. phagedenis. More exo- and endo-peptidase activities were found in T. denticola than in other species. The enteropathogenic T. hyodysenteriae isolates had a very low proteolytic profile. Dipeptidyl prolyl amidase activity was observed in all species except in the T. phagedenis Reiter strain and the avian intestinal spirochaetes. The enzyme profiles did not discriminate between oral and non-oral Treponema species.     

Genetic relatedness amongst intestinal spirochaetes isolated from rate and brids.

Multilocus enzyme electrophoresis was used to determine genetic relationships amongst 32 intestinal sprrochaetes ( Serpulina spp.) isolated from rats (17), rheas (7), chickens (4), ducks (2), a swan (1) and a flamingo (1). The strains were divided into 20 electrophoretic types (ETs), with a mean genetic diversity per locus of 0.62. The results were compared with those previously published for procine intestinal spirochaetes. One strain from a healthy rat, and three rhea strains which were recovered from cases of necrotizing typhlitis, were grouped in the same ETs as certain procine strains of Serpulina hydysenteriae . The rhea strains could be differentiated from these by pulsed-field gel electrophoresis. Fifteen of the rat strains could be differentiated from these by pulsed-field gel electrophoresis. Fifteen of the rat strains were genetically and phenotypically closely related. In contrast the avian strains were genetically more heterogeneous, with pathogenic isolates located in three different genetic groups.     

Detection of same sized 4.3 Kb extrachromosomal DNA elements in weakly beta-haemolytic human intestinal spirochaetes and Serpulina pilosicoli of swine origin

Agarose gel electrophoresis of total DNA from Italian strains of weakly beta-haemolytic human intestinal spirochaetes (w beta HIS) and porcine Serpulina pilosicoli reference strain P43/6/78 showed an extrachromosomal band having the same size and migrating at 4.3 Kb. The same results were observed after agarose gel electrophoresis of DNA obtained from the supernatant fluids of the spirochaetal cultures analysed. Swine Serpulina hyodysenteriae reference strain P18A was comparatively analysed and a 6.5 Kb extrachromosomal DNA element was found, as expected. Furthermore, S. hyodysenteriae reference strain P18A differed from all the other spirochaetes tested and had a higher number of flagella (8-12) at each cell end and was strongly beta-haemolytic. To the best of our knowledge, this is the first report on the detection of a band of extrachromosomal DNA having the same size in w beta HIS and S. pilosicoli from swine origin.     

Polymerase chain reaction for identification of human and porcine spirochaetes recovered from cases of intestinal spirochaetosis

Abstract A polymerase chain reaction (PCR) amplification of 16S rDNA was developed to identify spirochaetes recovered from cases of intestinal spirochaetosis in humans and pigs; these bacteria belong to a distinct genetic group of spirochaetes, with the proposed name ' Anguillina coll '. The PCR incorporated a universal eubacterial 16S rDNA sequencing primer (1492r), and a 21-base forward primer designed to include a nucleotide sequence specific for ' A. coli '. The PCR was used to correctly identify DNA extracted from 43 isolates of ' A. coli ' from humans and pigs, whilst no product was produced from Escherichia coli , or from other intestinal spirochaetes, including 38 isolates of Serpulina spp., and one each of Treponema succinifaciens and Brachyspira aalborgi . The amplification provided a rapid and simple means of identifying DNA from isolates of ' A. coli ', and could be used on boiled whole ' A. coli ' cells, with a detection limit equivalent to 2.5 × 10² cells. The reaction was used to detect and identify these spirochaetes from selective agar plates inoculated with stool specimens from infected pigs.     

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.     

Localization of carbohydrate and DNA in cyst-like structures from a human intestinal spirochaete

This study examined the cyst-like structures found in human intestinal spirochaetes by transmission electron microscopy and by histochemical and immunocytochemical analysis. A human intestinal spirochaete which morphologically resembled other intestinal spirochaetes was grown anaerobically on blood agar plates and in Tryptone Soya broth (Oxoid) and harvested by centrifugation after 8 days growth. Specimens were either conventionally fixed for transmission electron microscopy or fixed in 0.5% glutaraldehyde in 0.1M sodium cacodylate buffer and embedded in LR White resin for immunocytochemistry. En bloc histochemical investigation using a periodic acid-thiosemicarbazide-silver proteinate technique was undertaken for the localization of carbohydrate. A post-embedding immunogold labelling technique was used on ultrathin sections to label DNA. Results from the histochemical study demonstrated a reaction product which was confined to the cytoplasm of mature spirochaetes and in the central bodies within the cysts. Immunogold labelling demonstrated the presence of DNA in both the mature protoplasmic cylinders and in the central bodies. The results of the present study indicate that spirochaetal cysts are highly organized structures, which contain both DNA and carbohydrate. These findings are compatible with the view that these structures have a functional role rather than representing degenerative artifacts.     

Production and characterisation of a monoclonal antibody to Serpulina hyodysenteriae

Abstract A monoclonal antibody (mAb) directed against Serpulina hyodysenteriae , the causative agent of swine dysentery, was produced and characterised. The mAb (BJL/SH1) reacted in Western blots with a protein with a molecular mass of about 30 kDa in outer membrane preparations from a range of S. hyodysenteriae isolates of different serotypes. It did not react with preparations made from a variety of non- S. hyodysenteriae intestinal spirochaetes. Immunogold labelling was used to confirm the location of the reactive epitope on the cell outer membrane. The mAb agglutinated and produced fluorescence only with strains of S. hyodysenteriae , and should prove to be a useful reagent for identification of S. hyodysenteriae .     

Development and evaluation of polymerase chain reaction tests as an aid to diagnosis of swine dysentery and intestinal spirochaetosis

Polymerase chain reaction (PCR) tests were established for detection of Serpulina hyodysenteriae , the agent of swine dysentery, and S. pilosicoli , the agent of intestinal spirochaetosis. Both reactions were specific when tested with DNA from 107 strains of various intestinal spirochaetes. For diagnostic use, faeces were plated to selective medium, and diatomaceous earth extraction used to obtain DNA prior to PCR. This procedure detected 10³–10⁴ cells of either organism seeded into 0·2 g of faeces. When applied to 63 samples from pigs of eight piggeries naturally infected with either S. hyodysenteriae or S. pilosicoli , both PCRs were specific, more rapid, and detected more positive samples than did routine faecal culture and isolation.     

Variable small protein (Vsp)-dependent and Vsp-independent pathways for glycosaminoglycan recognition by relapsing fever spirochaetes

Tick-borne relapsing fever, caused by pathogenic Borrelia such as B. hermsii and B. turicatae, features recurrent episodes of bacteraemia, each of which is caused by a population of spirochaetes that expresses a different variable major protein. Relapsing fever is also associated with the infection of a variety of tissues, such as the central nervous system. In this study, we show that glycosaminoglycans (GAGs) mediate the attachment of relapsing fever spirochaetes to mammalian cells. B. hermsii strain DAH bound to immobilized heparin, and heparin and dermatan sulphate blocked bacterial binding to host cells. Bacterial binding was diminished by inhibition of host cell GAG synthesis or sulphation, or by the enzymatic removal of GAGs. GAGs mediated the attachment of relapsing fever spirochaetes to potentially relevant target cells, such as endothelial and glial cells. B. hermsii was able to attach to GAGs independently of variable major proteins, because strains expressing the variable major proteins Vsp33, Vlp7 or no variable major protein at all each recognized GAGs. Nevertheless, we found that a variable major protein of B. turicatae directly promoted GAG binding by this relapsing fever spirochaete. B. turicatae strain Oz1 serotype B, which expresses the variable major protein VspB, bound to GAGs more efficiently than did B. turicatae Oz1 serotype A, which expresses VspA. Recombinant VspB, but not VspA, bound to heparin and dermatan sulphate. Previous studies have shown that strain Oz1 serotype B grows to higher concentrations in the blood than does Oz1 serotype A. Thus, relapsing fever spirochaetes have the potential to express Vsp-dependent and Vsp-independent GAG-binding activities and, for one pair of highly related B. turicatae strains, differences in GAG binding correlate with differences in tissue tropism.     

Analysis of the axial filaments of Treponema hyodysenteriae by SDS-PAGE and immunoblotting

Purified axial filaments from eight serotypes of Treponema hyodysenteriae and two non-pathogenic intestinal spirochaetes were characterized by SDS-PAGE and Western blotting. Axial filaments of all ten strains had similar SDS-PAGE profiles; five major axial filament polypeptides were identified, with molecular masses of 43.8, 38, 34.8, 32.8 and 29.4 kDa. Hyperimmune gnotobiotic pig serum raised against purified axial filaments of strain P18A (serotype 4) cross-reacted with all other serotypes and with the non-pathogens, and convalescent serum taken from a pig with persistent swine dysentery also showed a strong response to the axial filament polypeptides. Hyperimmune gnotobiotic pig serum raised against axial filaments failed to agglutinate viable organisms and did not inhibit growth in vitro. Hence, the axial filaments of T. hyodysenteriae have been identified as major immunodominant antigens, although the role that antibodies to these antigens play in protection has yet to be established.     

The Lyme disease agent Borrelia burgdorferi requires BB0690, a Dps homologue, to persist within ticks

Borrelia burgdorferi survives in an enzootic cycle, and Dps proteins protect DNA against damage during starvation or oxidative stress. The role of a Dps homologue encoded by Borrelia in spirochaete survival was assessed. Dps-deficient spirochaetes were infectious in mice via needle-inoculation at the dose of 10(5) spirochaetes. Larval ticks successfully acquired Dps-deficient spirochaetes via a blood meal on mice. However, after extended periods within unfed nymphs, the Dps-deficient spirochaetes failed to be transmitted to a new host when nymphs fed. Our data suggest that Dps functions to protect the spirochaetes during dormancy in unfed ticks, and in its absence, the spirochaetes become susceptible during tick feeding. dps is differentially expressed in vivo- low in mice and high in ticks - but constitutively expressed in vitro, showing little change during growth or in response to oxidative stress. Borrelia Dps forms a dodecameric complex capable of sequestering iron. The Dps-deficient spirochaetes showed no defect in starvation and oxidative stress assays, perhaps due to the lack of iron in spirochaetes grown in vitro. Dps is critical for spirochaete persistence within ticks, and strategies to interfere with Dps could potentially reduce Borrelia populations in nature and thereby influence the incidence of Lyme disease.     

sodA is essential for virulence of Borrelia burgdorferi in the murine model of Lyme disease

Borrelia burgdorferi , the causative agent of Lyme disease, has a limited set of genes to combat oxidative/nitrosative stress encountered in its tick vector or mammalian hosts. We inactivated the gene encoding for superoxide dismutase A ( sodA , bb0153 ), an enzyme mediating the dismutation of superoxide anions and examined the in vitro and in vivo phenotype of the mutant. There were no significant differences in the in vitro growth characteristics of the sodA mutant compared with the control strains. Microscopic analysis of viability of spirochaetes revealed greater percentage of cell death upon treatment of sodA mutant with superoxide generators compared with its controls. Infectivity analysis in C3H/HeN mice following intradermal needle inoculation of 10³ or 10⁵ spirochaetes per mouse revealed complete attenuation of infectivity for the sodA mutant compared with control strains at 21 days post infection. The sodA mutant was more susceptible to the effects of activated macrophages and neutrophils, suggesting that its in vivo phenotype is partly due to the killing effects of activated immune cells. These studies indicate that SodA plays an important role in combating oxidative stress and is essential for the colonization and dissemination of B. burgdorferi in the murine model of Lyme disease.     

From tooth to hoof: treponemes in tissue-destructive diseases

With the advent of new molecular and immunological tools, there is better understanding of the roles that difficult to cultivate bacteria, and not-yet-cultivated bacteria such as spirochaetes, play in polymicrobial diseases. Only relatively recently have studies implicated Treponema spirochaetes in human periodontal disease, a destructive condition of the tissues supporting the teeth. A number of different Treponema species have been isolated and their surface protein components that mediate adhesion, cytotoxicity, and tissue damage have been characterized. More recently Treponema strains closely related to human oral isolates have been cultivated from active lesions of digital dermatitis, an ulcerative condition affecting the feet of cows and sheep. This condition, like periodontal disease, appears to have a polymicrobial aetiology in which enrichment for Treponema may play a crucial part. This article reviews the known mechanisms by which Treponema interact with eukaryotic host cells and tissue proteins, and how these interactions may contribute to pathogenic diversity.