The Spirochete Brachyspira pilosicoli, Enteric Pathogen of Animals and Humans.

Brachyspira pilosicoli is a slow-growing anaerobic spirochete that colonizes the large intestine. Colonization occurs commonly in pigs and adult chickens, causing colitis/typhlitis, diarrhea, poor growth rates, and reduced production. Colonization of humans also is common in some populations (individuals living in village and peri-urban settings in developing countries, recent immigrants from developing countries, homosexual males, and HIV-positive patients), but the spirochete rarely is investigated as a potential human enteric pathogen. In part this is due to its slow growth and specialized growth requirements, meaning that it is not detectable in human fecal samples using routine diagnostic methods. Nevertheless, it has been identified histologically attached to the colon and rectum in patients with conditions such as chronic diarrhea, rectal bleeding, and/or nonspecific abdominal discomfort, and one survey of Australian Aboriginal children showed that colonization was significantly associated with failure to thrive. B. pilosicoli has been detected in the bloodstream of elderly patients or individuals with chronic conditions such as alcoholism and malignancies. This review describes the spirochete and associated diseases. It aims to encourage clinicians and clinical microbiologists to consider B. pilosicoli in their differential diagnoses and to develop and use appropriate diagnostic protocols to identify the spirochete in clinical specimens.

Vaccination of chickens with the 34†kDa carboxy-terminus of Bpmp72 reduces colonization with Brachyspira pilosicoli following experimental infection.

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of a variety of species of mammals and birds, and may result in colitis, diarrhoea and reductions in growth rate. Naturally occurring infections in chickens are largely confined to adult laying and breeding birds. In this study, the 34 kD carboxy-terminus of the prominent outer membrane protein Bmp72 of B. pilosicoli was expressed as a histidine-tagged recombinant protein and used to immunize two groups (B and C) of 15 individually housed layer chickens. Vaccination was with either 100 µg (B) or 1 mg (C) protein emulsified with Freund's incomplete adjuvant delivered into the pectoral muscles, followed three weeks later by 1 mg of protein in phosphate buffered saline delivered via crop tube. Two weeks later these and 15 non-vaccinated positive control birds (group A) housed in the same room were challenged via crop tube with B. pilosicoli avian strain CPS1. B. pilosicoli was detected in the faeces of all control birds and in 14 of the vaccinated birds in each vaccinated group at some point over the 30-day period following challenge. Colonization was delayed and the duration of excretion was significantly reduced (P = 0.0001) in both groups of vaccinated birds compared to the non-vaccinated control birds. Fewer immunized birds had abnormal caecal contents at post mortem examination compared to non-vaccinated birds, but the difference was not statistically significant. This study indicates that recombinant Bmp72 C-terminus has potential to be developed for use as a vaccine component to provide protection against B. pilosicoli infections. RESEARCH HIGHLIGHTS Laying chickens were immunized with recombinant Brachyspira pilosicoli membrane protein Bpmp72. Immunized birds had a highly significant reduction in the duration of colonization. Fewer immunized than control birds had abnormal caecal contents after infection. Bpmp72 showed potential for use as a novel vaccine component for B. pilosicoli.

Brachyspira suanatina sp. nov., an enteropathogenic intestinal spirochaete isolated from pigs and mallards: genomic and phenotypic characteristics.

BACKGROUND: The genus Brachyspira currently encompasses seven valid species that colonize the intestines of mammals and birds. In a previous study a group of strongly haemolytic isolates from pigs and mallards was provisionally described as a new species within genus Brachyspira, "B. suanatina", and enteropathogenic properties were demonstrated in a porcine challenge model. METHODS: In the current study characterization of B. suanatina was performed on the basis of cell morphology, growth characteristics, enzyme profiles, DNA-DNA hybridization (DDH) and whole genome comparisons. The draft genome sequence of B. suanatina strain AN4859/03 was determined and compared with the available genomes of all valid species of Brachyspira. RESULTS: According to morphological traits, growth characteristics and enzymatic profiles, B. suanatina was similar to the type strain of B. hyodysenteriae, but using the recommended threshold value of 70% similarity by DDH it did not belong to any of the recognized Brachyspira species (range 16-64% similarity). This was further supported by average nucleotide identity values. Phylogenetic analysis performed using housekeeping genes and core genomes of all valid Brachyspira sp. and "B. hampsonii" revealed that B. suanatina and B. intermedia formed a clade distinct from B. hyodysenteriae. By comparing the genomes of the three closely related species B. intermedia, B. hyodysenteriae and B. suanatina similar profiles of general genomic features and distribution of genes in different functional categories were obtained. However, the genome size of B. hyodysenteriae was smallest among the species, suggesting the possibility of reductive evolution in the divergence of this species. A bacteriophage region and a putative plasmid sequence were also found in the genome of B. suanatina strain AN4859/03. CONCLUSIONS: The results of our study suggest that despite being similar to B. hyodysenteriae phenotypically, B. suanatina should be regarded as a separate species based on its genetic characteristics. Based on characteristics presented in this report we propose that strains AN4859/03, AN1681:1/04, AN2384/04 and Dk12570-2 from pigs in Sweden and Denmark, and strains AN3949:2/02 and AN1418:2/01 isolated from mallards in Sweden, represent a unique species within genus Brachyspira. For this new species we propose the name B. suanatina for which the type strain is AN4859/03T (=ATCC® BAA-2592™=DSM 100974T).

Strains of the intestinal spirochaete Brachyspira pilosicoli attach to and aggregate erythrocytes.

UNLABELLED: The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of various species of mammals and birds, where it may induce colitis. Strains of the spirochaete have also been isolated from the bloodstream of immunocompromised human patients and have been seen in liver sections, and a similar systemic spread was recently observed in experimentally infected chickens. Some other spirochaete species that may be present in blood attach to and aggregate erythrocytes, and this is believed to contribute to disease severity. The aim of the current study was to determine whether B. pilosicoli strains have the capacity to attach to and aggregate erythrocytes. Initially, four strains of B. pilosicoli were incubated with erythrocytes from sheep, cows, pigs, dogs, humans, chickens and geese, and were observed by phase-contrast microscopy. Only strain WesB attached, and this was only with erythrocytes from chickens and geese. Subsequently, six other strains of B. pilosicoli were tested just with goose erythrocytes, and five attached to and caused aggregation of the erythrocytes. Scanning and transmission electron microscopy demonstrated that spirochaetes abutted and apparently firmly attached to the erythrocyte membranes. Aggregation of erythrocytes by B. pilosicoli may contribute to disease severity in species that develop a spirochaetaemia. SIGNIFICANCE AND IMPACT OF THE STUDY: The intestinal spirochaete Brachyspira pilosicoli has been isolated from the bloodstream of immunocompromised human patients, and spread to the liver has been reported in humans and in experimentally infected chickens. In this study, B. pilosicoli was shown to undergo attachment by one cell end to chicken and goose erythrocytes in vitro and to aggregate them. This activity has the potential to contribute to disease severity in avian and possibly other species that develop a spirochaetaemia and systemic spread. Avian erythrocytes may be useful for studying the mechanisms by which B. pilosicoli attaches to cells.

Fecal shedding of Brachyspira spp. on a farrow-to-finish swine farm with a clinical history of "Brachyspira hampsonii"-associated colitis.

BACKGROUND: Brachyspira associated diarrhea is a re-emerging concern for Canadian swine producers. To identify critical control points for reducing the impact of Brachyspira on production, improved diagnostic tools and a better understanding of the on-farm epidemiology of these pathogens are required. A cross-sectional study was conducted for the detection of Brachyspira on a commercial, two-site, farrow-to-finish pork production unit in Saskatchewan, Canada with a clinical history of mucohaemorrhagic colitis associated with "B. hampsonii". RESULTS: Rectal swabs from pigs at all production stages were collected over 13 weeks (n=866). Two swabs were collected per pig for culture and Gram stain, and for PCR. Ninety-one culture positive samples were detected, with the highest prevalence of Brachyspira shedding in grower pigs (21%). No Brachyspira were detected in pre-weaned piglets. PCR and Gram stain of rectal swabs detected fewer positive samples than culture. The most prevalent species detected was B. murdochii; other species detected included B. pilosicoli, B. innocens, and "Brachyspira hampsonii". Phylogenetic analysis revealed that several of the isolates, including some strongly beta-haemolytic isolates, might represent novel taxa. CONCLUSIONS: Our results indicate that apparently healthy pigs can be colonized with diverse Brachyspira species, including some potential pathogens, and that frequency of shedding peaks in the grower stage. Difference in the detection rates of Brachyspira amongst culture, Gram stain or PCR on rectal swabs have implications for choice of detection methods and surveillance approaches that may be most effective in Brachyspira control strategies.

Attraction of Brachyspira pilosicoli to mucin.

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of various species, including humans. In the colon this spirochaete can penetrate the overlying mucus layer, attach by one cell end to the underlying enterocytes, and initiate localized colitis and diarrhoea. The aim of this study was to investigate whether, as part of the colonization process, B. pilosicoli is attracted to mucin. Fifteen B. pilosicoli strains isolated from humans, pigs, chickens and dogs, and a control strain of Brachyspira hyodysenteriae, were analysed for their ability to enter solutions of hog gastric mucin in an in vitro capillary tube assay. No significant attraction was detected with 1 % mucin, but some strains started to enter a 2 % solution, and attraction then increased with increasing concentrations to peak at around 6-8 % mucin. A similar increase was seen with B. hyodysenteriae, although this activity peaked at 6 % mucin and then declined, suggesting that the two species have different affinities for mucin. These mucin concentrations were much higher than those used in previous experimental studies with Brachyspira species. The viscosities of the 6-8 % mucin solutions were around 7-12 mPa s, which were similar to the measured viscosities of the mucus layer overlying the epithelium in the caecum and colon of experimental pigs. The strains varied in their motility, as assessed by their ability to enter tubes containing chemotaxis buffer, but there was no significant relationship between this motility and the extent of their ability to enter the mucin solutions. Different strains also had different propensities to enter the mucin solutions, but there were no consistent differences according to the host species of origin. B. pilosicoli strain 95/1000 was attracted towards a solution of d-serine, suggesting that chemotaxis was involved in the attraction to mucin; however, 95/1000 was also attracted to viscous solutions of polyvinylpyrrolidone (PVP), in a manner mirroring the response to mucin, and hence suggesting the involvement of viscotaxis in the attraction to mucin. B. hyodysenteriae B204 showed a similar viscotaxis to PVP. Further studies are required to determine whether the in vitro interaction of a given strain with mucin is a useful indicator of its in vivo colonization ability, and hence could be used as a potential marker for virulence.

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.

Intestinal spirochetes isolated from wild-living jackdaws, hooded crows and rooks (genus Corvus): provisionally designated "Brachyspira corvi" sp. nov.

Intestinal spirochetes of genus Brachyspira are commonly isolated from mammalian and avian hosts, and several species have been reported to cause enteric disease in pigs and birds. Except for a previous publication on three isolates from corvid birds (order Passeriformes, family Corvidae, genus Corvus), of which two are further studied in this paper, no other reports exist on Brachyspira spp. of passerine birds. In this study, cloacal and intestinal swabs of small and large intestines were collected from 116 corvid birds of three species, i.e. jackdaws (Corvus monedula), hooded crows (Corvus corone cornix) and rooks (Corvus frugilegus), from four separate geographical locations in Sweden. Isolates were obtained by selective culture from 43 of 116 birds. All isolates were weakly hemolytic, indole-negative and lacked hippurate cleavage capacity. Examination by light microscopy did not indicate association with enteric disease in necropsied birds. Pure spirochete cultures were obtained by serial dilution and subculture, and selected isolates were analyzed by PCR (n=14), randomly amplified polymorphic DNA (RAPD) (n=14), and sequencing of the almost complete 16S rRNA (n=14), and partial nox genes (n=4). Positive reactions were noticed by PCR targeting a hexa-T segment of the 16S rRNA gene, which has been previously reported as a signature characteristic of Brachyspira pilosicoli. By 16S rRNA gene sequencing, the isolates formed a separate cluster related to genus Brachyspira, but not consistent with any presently recognized or proposed Brachyspira sp. The sequence similarity of the 16S rRNA gene among the isolates from corvid birds was 99.7-100%. Compared to 16S rRNA gene sequence data from all presently recognized and several proposed Brachyspira spp. the sequence similarity of the isolates from corvid birds varied between 94.1 and 96.5%. In a radial tree based on nox gene sequences, all four analyzed isolates from corvid birds formed a separate cluster. By RAPD analysis, the banding patterns of the isolates differed from all type strains of Brachyspira spp. Based on the results presented in this paper, we propose that the described isolates from corvid birds belong to a novel species within genus Brachyspira, with the provisional name "Brachyspira corvi" (cor'vi. L gen. n. corvi, of a crow).

Infection of porcine colon explants with "Brachyspira hampsonii" leads to increased epithelial necrosis and catarrhal exudate.

Mucohemorrhagic diarrhea in pigs caused by Brachyspira spp. has a global distribution, and an economic impact on affected farms due to poor performance of animals. Demonstrations that "Brachyspira hampsonii" is pathogenic have been achieved using in vivo animal models, but a critical knowledge gap exists regarding the pathogenic mechanisms employed by Brachyspira. Here, we used in vitro organ culture of porcine colon to investigate interactions between "B. hampsonii" and explants during the first 12 h of contact. Explants were either inoculated with "B. hampsonii" or sterile culture broth. Responses to infection were evaluated by optical microscopy and quantitative PCR. Significantly greater numbers of necrotic crypt cells and thicker catarrhal exudate were observed on infected explants compared to controls. Spirochaetes were observed in the mucus layer, in contact with necrotic exfoliated cells, in crypts and the lamina propria. Statistical differences were observed in mRNA levels between inoculated and control explants for IL-1α, TNF-α and ZO-1 using a Bayesian analysis, but not observed using the ΔΔCq method. These results provide a demonstration of a porcine colon explant model for investigating interactions of Brachyspira with its host and show that initial effects on the host are observed within the first 12 h of contact.

Experimental infection of conventional pigs with a 'Brachyspira hampsonii' isolate recovered from a migrating waterfowl in Spain.

'Brachyspira hampsonii' is a recently proposed new species within the Brachyspira genus, which produces a dysentery-like disease in pigs. This study aims at investigating whether a 'B. hampsonii' isolate recovered from a migrating waterfowl was capable of colonizing pig intestines, inducing clinical signs of dysentery and being transmitted among pigs. Eleven 7-week-old pigs were randomly assigned into two separate groups which were orally administered an avian isolate of 'B. hampsonii' (inoculated group, n = 5) or BHI broth (control group, n = 6). After inoculation, three pigs from the control group were placed in the inoculated pen and served as sentinel pigs. Our results show the capacity of this avian 'B. hampsonii' isolate to colonize the large intestine of pigs and to be transmitted among pigs. According to this, migrating birds could play a role in the epidemiology of 'B. hampsonii' as a possible source of infection in swine populations.

Spirochete chemotaxis, motility, and the structure of the spirochetal periplasmic flagella.

Spirochetes have a unique motility system that is characterized by flagellar filaments contained within the outer membrane sheath. Direct evidence using video microscopy has recently been obtained which indicates that these periplasmic flagella (PF) rotate in several spirochetal species. This rotation generates thrust. As shown for one spirochete, Spirochaeta aurantia, motility is driven by a proton motive force. Spirochete chemotaxis has been most thoroughly studied in S. aurantia. This spirochete exhibits three distinct behaviours, runs of smooth swimming, reversals and flexing. These behaviours are modulated by addition of attractants such that S. aurantia swims towards higher concentrations of attractants in a spatial gradient. Unlike the prototypical bacterium, Escherichia coli, chemotaxis in S. aurantia involves fluctuations in membrane potential. The PF of a number of spirochetes have been examined in considerable detail. For most species, the PF filaments are complex, consisting of an assembly of several different polypeptides. There are several antigenically related core polypeptides surrounded by an outer layer consisting of a different polypeptide. Borrelia burgdorferi and Spirochaeta zuelzerae represent exceptions where the filaments are composed of a single major polypeptide species. The genes encoding the filament polypeptides from several spirochete species have been cloned and analysed. Apparently, the outer layer polypeptides of S. aurantia, Treponema pallidum and Serpulina hyodysenteriae are transcribed from sigma-70-like promoters, whereas the core polypeptide genes are transcribed from sigma-28-like promoters. A gene encoding the hook polypeptide in Treponema phagedenis has been cloned and analysed. The product of this gene shows significant similarity to the E. coli hook protein, FlgE, and homologs have been identified in T. pallidum and B. burgdorferi.(ABSTRACT TRUNCATED AT 250 WORDS)

Examination of Serpulina pilosicoli for attachment and invasion determinants of Enterobacteria.

The spirochaete, Serpulina pilosicoli, is the agent of intestinal spirochaetosis, a diarrhoeal disease of humans and other species. By mechanisms as yet unknown, large numbers of these spirochaetes intimately attach to the colonic mucosa by one cell end. In some infected individuals, the spirochaetes may invade the lamina propria and adjacent tissues, and they may cause spirochaetaemia. To examine S. pilosicoli for pathogenic determinants homologous with Enterobacteria, DNA was extracted from six strains of S. pilosicoli and hybridised at low stringency with DNA probes derived from the inv, ail and yadA genes of Yersinia enterocolitica, the eae gene from enteropathogenic Escherichia coli and a probe derived from the virulence plasmid of Shigella flexneri. No hybridisation of the enterobacterial probes to S. pilosicoli DNA was detected, indicating that these gene sequences, which are known to be involved in the attachment and invasion processes of the other intestinal pathogens, were not present in the spirochaetes.

Intestinal spirochete infections of chickens: a review of disease associations, epidemiology and control.

This paper presents an overview of intestinal spirochete infections of chickens. It focuses particularly on studies in Australia, where recent surveys of 136 layer and broiler breeder flocks have revealed a high rate of infection (>40%) with intestinal spirochetes. Infection was not detected in broiler flocks. Approximately 50% of isolates from infected flocks were Brachyspira (Serpulina) intermedia or B. pilosicoli, with the other isolates being B. innocens, B. murdochii or the proposed species 'B. pulli'. No isolates of B. alvinipulli were found. Intestinal spirochetes were significantly associated with wet litter problems and/or reduced egg production. Experimental infection of point-of-lay birds with either B. intermedia or B. pilosicoli caused reduced egg production, and, with B. intermedia, a significant increase in fecal moisture content. Infection with B. innocens caused no significant changes. In-water treatment of a flock with a mixed spirochete infection using lincospectin resulted in a slimy diarrhea lasting for 2-3 weeks, followed by absence of spirochetes for 3 months. Birds treated with tiamulin remained healthy, and had a reduced level of infection with intestinal spirochetes (30%) for 3 months. Trials are under way to test the efficacy of antimicrobials in point-of-lay chickens experimentally infected with either B. intermedia or B. pilosicoli.

Motility-regulated mucin association of Serpulina pilosicoli, the agent of colonic spirochetosis of humans and animals.

Colonic spirochetosis is a disease of humans and animals characterized by colonization of the colonic mucus gel and intimate attachment of Serpulina pilosicoli to the apical membrane of enterocytes. Motility-regulated mucin association plays a key role in colonic infection by the related spirochete Serpulina hyodysenteriae, the cause of swine dysentery. In this study the chemotaxis of Serpulina pilosicoli porcine isolate P43/6/78, human isolate SP16, and canine isolate 16242-94 was examined by anaerobic incubation of each spirochete in control medium or medium containing increasing concentrations of D-L serine or porcine gastric mucin (PGM). The porcine isolate had a chemotactic response towards 10 mM D-L serine, but not towards PGM. By contrast, the human and canine isolates were attracted towards 0.1% PGM, but not towards DL-serine. The composition of the growth medium appeared to modulate the chemotactic response of S. pilosicoli towards PGM; the loss of a chemotactic response of spirochetes grown in medium without pig fecal extract was restored by growing the spirochetes in medium containing 0.1% PGM. Serpulina pilosicoli displays a chemotactic response towards PGM which is modulated by the presence of certain substrate during the growth phase of the spirochete.

Characterization of the fecal microbiota of pigs before and after inoculation with "Brachyspira hampsonii".

"Brachyspira hampsonii" causes disease indistinguishable from swine dysentery, and the structure of the intestinal microbiome likely plays a role in determining susceptibility of individual pigs to infection and development of clinical disease. The objectives of the current study were to determine if the pre-inoculation fecal microbiota differed between inoculated pigs that did (INOC MH) or did not (INOC non-MH) develop mucohaemorrhagic diarrhea following challenge with "B. hampsonii", and to quantify changes in the structure of the microbiome following development of clinical disease. Fecal microbiota profiles were generated based on amplification and sequencing of the cpn60 universal target sequence from 89 samples from 18 pigs collected at -8, -5, -3 and 0 days post-inoculation, and at termination. No significant differences in richness, diversity or taxonomic composition distinguished the pre-inoculation microbiomes of INOC MH and INOC non-MH pigs. However, the development of bloody diarrhea in inoculated pigs was associated with perturbation of the microbiota relative to INOC non-MH or sham-inoculated control pigs. Specifically, the fecal microbiota of INOC MH pigs was less dense (fewer total 16S rRNA copies per gram of feces), and had a lower Bacteroidetes:Firmicutes ratio. Further investigation of the potential long-term effects of Brachyspira disease on intestinal health and performance is warranted.

Reproduction of mucohaemorrhagic diarrhea and colitis indistinguishable from swine dysentery following experimental inoculation with "Brachyspira hampsonii" strain 30446.

BACKGROUND: Mucohaemorrhagic diarrhea caused by Brachyspira hyodysenteriae, swine dysentery, is a severe production limiting disease of swine. Recently, pigs in western Canada with clinical signs indistinguishable from swine dysentery were observed. Despite the presence of spirochetes on fecal smears, recognized Brachyspira spp. including B. hyodysenteriae could not be identified. A phylogenetically distinct Brachyspira, called "B. hampsonii" strain 30446, however was isolated. The purpose of this study was to experimentally reproduce mucohaemorrhagic colitis and characterize strain 30446 shedding following inoculation. METHODS AND FINDINGS: Eighteen 13-week-old pigs were randomly assigned to inoculation (n = 12) or control (n = 6) groups in each of two trials. In trial 1, pigs were inoculated with a tissue homogenate collected from clinically affected field cases. In trial 2, pigs were inoculated with a pure broth culture of strain 30446. In both trials, mucohaemorrhagic diarrhea was significantly more common in inoculated pigs than controls, all of which remained healthy. In animals with mucohaemorrhagic diarrhea, significantly more spirochetes were observed on Gram stained fecal smears, and higher numbers of strain 30446 genome equivalents were detected by quantitative PCR (qPCR). Strain 30446 was cultured from colon and/or feces of all affected but no control animals at necropsy. CONCLUSIONS: "Brachyspira hampsonii" strain 30446 causes mucohaemorrhagic diarrhea in pigs following a 4-9 day incubation period. Fecal shedding was detectable by day 4 post inoculation, and rarely preceded the onset of mucoid or haemorrhagic diarrhea by more than 2 days. Culture and 30446-specific qPCR are reliable methods of detection of this organism in feces and tissues of diarrheic pigs. The emergence of a novel Brachyspira spp., such as "B. hampsonii", creates diagnostic challenges including higher risk of false negative diagnostic tests. We therefore recommend diagnostic laboratories routinely use Brachyspira culture, nox-based and species-specific PCR, and DNA sequencing to diagnose Brachyspira-associated colitis in pigs.

The histological features of intestinal spirochetosis in a series of 113 patients.

A multicenter retrospective review of cases diagnosed as spirochetosis was performed to determine the presenting symptoms and histological changes. A total of 113 cases were retrieved from the archives, consisting of 97 colorectal specimens and 16 appendices. In only 25 cases was the presenting symptom recorded as diarrhea. Of the colorectal specimens, 87 (90%) showed no mucosal abnormality (apart from the spirochetes); the other 10 showed mucosal inflammation but 6 of them had a diagnosis of another inflammatory disease process accounting for the inflammatory changes. Five appendices showed acute appendicitis; the other 11 were unremarkable. It is concluded that spirochetosis in an unselected general population is unlikely to be of pathological significance. Furthermore, if spirochetosis is observed in an inflamed biopsy, it is most likely to be an incidental finding.

Prevalence of Brachyspira pilosicoli and "Brachyspira canis" in dogs and their association with diarrhoea.

The aims of this study were to investigate the prevalence of colonization with intestinal spirochaetes in dogs, and to assess their association with diarrhoea. To achieve this, faecal samples from 311 dogs were obtained between November 2008 and April 2009 and cultured for Brachyspira species. A total of 41 Brachyspira spp. isolates were recovered, and these were classified into species according to their biochemical properties, and results of a B. pilosicoli species-specific PCR, and partial amplification of the nox gene with sequencing of the product. An overall Brachyspira spp. prevalence of 13.2% (41/311) was obtained. The prevalence of Brachyspira pilosicoli faecal shedding was 4.8% (15/311) while "Brachyspira canis" was identified in 8.0% (25/311) of the sampled dogs. One dog shed an isolate tentatively identified as B. intermedia. A statistically significant association between the shedding of B. pilosicoli and the presence of diarrhoea in dogs was demonstrated (P<0.001). Risk factors for shedding of Brachyspira spp. were investigated. Using the odds ratio, the risk of B. pilosicoli shedding was five times higher among dogs up to 1 year of age as compared with adult dogs (older than 1 year). These findings may have practical implications in the public and animal health fields.

In vitro antagonistic activities of Lactobacillus spp. against Brachyspira hyodysenteriae and Brachyspira pilosicoli.

The sensitivity of Brachyspira hyodysenteriae and Brachyspira pilosicoli, respectively the causative agents of Swine Dysentery and Porcine Intestinal Spirochaetosis to two probiotic Lactobacillus strains, L. rhamnosus CNCM-I-3698 and L. farciminis CNCM-I-3699 was studied through viability, motility and coaggregation assays. The cell-free supernatant of these lactobacilli contains lactic acid, that is stressful for Brachyspira (leading to the formation of spherical bodies), and lethal. It was demonstrated for the first time the in vitro coaggregation properties of two probiotic Lactobacillus strains (active or heat-treated) with two pathogenic strains of Brachyspira, leading to (1) trapping of spirochaetal cells in a physical network as demonstrated by SEM; (2) inhibition of the motility of Brachyspira. Such in vitro studies should encourage in vivo studies in animal model to evaluate the potential of the use of probiotic lactobacilli through a feeding strategy for the prevention of B. hyodysenteriae and B. pilosicoli.

Reclassification of Serpulina intermedia and Serpulina murdochii in the genus Brachyspira as Brachyspira intermedia comb. nov. and Brachyspira murdochii comb. nov.

A reassessment was undertaken of published characteristics of the five species of anaerobic intestinal spirochaetes in the genus Brachyspira (Brachyspira aalborgi, Brachyspira alvinipulli, Brachyspira hyodysenteriae, Brachyspira innocens and Brachyspira pilosicoli) and the two species in the genus Serpulina (Serpulina intermedia and Serpulina murdochii). Comparisons were made of published descriptions, including phenotypic properties, grouping by multilocus enzyme electrophoresis, DNA base composition, DNA-DNA relative reassociation values and 16S rRNA gene sequence similarity. On the basis of extensive similarities between all the species, it is proposed to reclassify Serpulina intermedia Stanton et al. 1997 and Serpulina murdochii Stanton et al. 1997 in the genus Brachyspira, as Brachyspira intermedia comb. nov. (type strain ATCC 51140T) and Brachyspira murdochii comb. nov. (type strain ATCC 51284T).