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Brachyspira hyodysenteriae is commonly associated with swine dysentery (SD), a disease that has an economic impact on the swine industry. B. hyodysenteriae infection results in changes to the colonic mucus niche with massive mucus induction, which substantially increases the number of B. hyodysenteriae binding sites in the mucus. We previously determined that a B. hyodysenteriae strain binds to colon mucins in a manner that differs between pigs and mucin types. Here, we investigated if adhesion to mucins is a trait observed across a broad set of B. hyodysenteriae strains and isolates and furthermore at a genus level ( B. innocens , B. pilosicoli , B. murdochii , B. hampsonii , and B. intermedia strains). Our results show that binding to mucins appears to be specific to B. hyodysenteriae, and within this species, the binding ability to mucins varies between strains/isolates, increases for mucins from pigs with SD, and is associated with sialic acid epitopes on mucins. Infection with B. hyodysenteriae strain 8dII results in mucin glycosylation changes in the colon, including a shift in sialic acid-containing structures. Thus, we demonstrate through hierarchical cluster analysis and orthogonal projections to latent structures discriminant analysis (OPLS-DA) models of the relative abundances of sialic acid-containing glycans that sialic acid-containing structures in the mucin O -glycome are good predictors of B. hyodysenteriae strain 8dII infection in pigs. The results emphasize the role of sialic acids in governing B. hyodysenteriae interactions with its host, which may open perspectives for therapeutic strategies.

A novel TaqMan 5-plex real-time PCR using a combination of locked nucleic acid-modified (LNA)- and minor groove binding (MGB)-conjugated DNA probes was developed for identification and differentiation between the four main pathogenic Brachyspira species in swine. B. hyodysenteriae, B. pilosicoli, and B. suanatina are identified using three hydrolysis probes targeting cpn60, while B. hampsonii is recognized by another nox specific probe. The assay also includes an exogenous internal control simultaneously verifying the PCR competency of the DNA samples. Validation of the novel assay was performed using DNA samples from 18 Brachyspira reference strains and 477 clinical samples obtained from porcine rectal swabs by comparing them with different PCR-based methods targeting nox, 16S rDNA, and 23S rDNA. The specificity of the assay was 100% without cross-reactivity or detection of different pathogens. Depending on the Brachyspira species, the limit of detection was between 10 and 20 genome equivalents with a cut-off threshold cycle (Ct) value of 37. The developed highly sensitive and specific 5-plex real-time PCR assay is easy to implement in routine veterinary diagnostic laboratories and enables rapid differentiation between the main four pathogenic Brachyspira species recognized in pigs using a single-tube approach., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

The enteric, pathogenic spirochaete Brachyspira pilosicoli colonizes and infects a variety of birds and mammals, including humans. However, there is a paucity of genomic data available for this organism. This study introduces 12 newly sequenced draft genome assemblies, boosting the cohort of examined isolates by fourfold and cataloguing the intraspecific genomic diversity of the organism more comprehensively. We used several in silico techniques to define a core genome of 1751 genes and qualitatively and quantitatively examined the intraspecific species boundary using phylogenetic analysis and average nucleotide identity, before contextualizing this diversity against other members of the genus Brachyspira . Our study revealed that an additional isolate that was unable to be species typed against any other Brachyspira lacked putative virulence factors present in all other isolates. Finally, we quantified that homologous recombination has as great an effect on the evolution of the core genome of the B. pilosicoli as random mutation ( r / m =1.02). Comparative genomics has informed Brachyspira diversity, population structure, host specificity and virulence. The data presented here can be used to contribute to developing advanced screening methods, diagnostic assays and prophylactic vaccines against this zoonotic pathogen.

The pig colon is the habitat of diverse Brachyspira species, of which only a few are of clinical importance. Methods for identification have shifted from phenotypic to molecular testing over the last two decades. Following the emergence of B. hampsonii it became evident that relying on species-specific PCRs carries the risk of overlooking important new species. Consequently, sequencing was proposed as an unbiased alternative for identification of isolates. So far, the main target for identification across species has been the NADH oxidase gene (nox). However, multiple copies of this gene in the genome and potential lateral gene transfer reduce confidence when using this gene. This study compared identification and phylogentic relationship inferred from nox sequencing to that inferred from sequencing of the cpn60 universal target using a collection of 168 isolates from different Brachyspira species. The majority of isolates had an identical identification with both methods. There were a few outliers in the trees with uncertain assignment to a species by BLAST analysis. A few major discrepancies pertained to the pathogenic species B. hampsonii (2), B. pilosicoli (1) and B. suanatina (1). Weakly haemolytic variants of B. hyodysenteriae were assigned to the correct species by both methods. Some of the isolates identified as B. hampsonii also had a weakly haemolytic phenotype., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To date nine species of anaerobic intestinal spirochaetes have been validly assigned to the genus Brachyspira. These include both pathogenic and non-pathogenic species. In the current study a genomic analysis of a novel spirochaete isolate was undertaken to determine whether it is a distinct species that previously has been misidentified as Brachyspira aalborgi. The genome of spirochaete strain Z12 isolated from the faeces of a vervet monkey was sequenced and compared to the genomes of the type strains of the nine assigned Brachyspira species. Genome to Genome Distance (GGD) values and Average Nucleotide Identity (ANI) values were determined. Single nucleotide polymorphisms (SNP) were used to create a phylogenetic tree to assess relatedness. The 16S rRNA gene sequences of the strains were aligned and the similarity amongst the Brachyspira species was recorded. Multilocus sequence typing (MLST) using five loci was conducted on Z12 and results compared with those for other Brachyspira isolates. Assembly of the Z12 sequences revealed a 2,629,108 bp genome with an average G + C content of 31.3%. The GGD, ANI, 16S rRNA gene sequence comparisons and the MLST results all indicated that Z12 represents a distinct species within the genus Brachyspira, with its nearest neighbour being B. aalborgi. Spirochaete strain Z12 T was assigned as the type strain of a new species, Brachyspira catarrhinii sp. nov. The diagnostic PCR currently in use to detect B. aalborgi cross-reacts with Z12, but RFLP analysis of PCR product can be used to distinguish the two species. Previous reports of non-human primates being colonised by B. aalborgi based on PCR results may have been incorrect. The development of an improved diagnostic method will allow future studies on the distribution and possible clinical significance of these two anaerobic spirochaete species., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Avian intestinal spirochetosis (AIS), an important but neglected disease in laying hens, is caused by Brachyspira pilosicoli, B. intermedia, and B. alvinipulli. Poultry are also frequently colonized by putatively nonpathogenic species such as B. murdochii and B. innocens. We evaluated the differentiation of Brachyspira species by 3 methods: sequencing of the reduced nicotinamide adenine dinucleotide (NADH) oxidase gene ( nox), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and a new multiplex (m)PCR targeting genes such as the tryptophanase A gene ( tnaA) and the p-aminobenzoyl-glutamate hydrolase subunit B gene ( abgB). Sequencing of 414 bp of the nox PCR amplification products generated from 41 pure cultures of avian Brachyspira isolates allowed presumptive species identification in 33 isolates with at least 99% identity in basic local alignment search tool analysis, including B. pilosicoli, B. intermedia, B. murdochii, B. innocens, and " B. pulli". MALDI-TOF MS analysis was found to be a reliable tool for differentiation after extension of the manufacturer's database. In the mPCR, all isolates identified as B. pilosicoli and B. intermedia were positive for abgB and tnaA, respectively. The mPCR might be very useful in detecting Brachyspira species in mixed cultures including not only nonpathogenic species, such as B. innocens, but also one of the AIS pathogens. We found that MALDI-TOF MS analysis combined with the mPCR targeting tnaA and abgB was suitable for the identification of avian isolates of B. pilosicoli and B. intermedia, 2 important agents of AIS.

The 'colonic' spirochetes assigned to the genus Brachyspira are slow-growing anaerobic bacteria. The genus includes both pathogenic and non-pathogenic species, and these variously colonise the large intestines of different species of birds and animals, including humans. Scientific understanding of the physiology and molecular biology of Brachyspira spp. remains very limited compared with that of other pathogenic spirochetes, and there are few descriptions of successful genetic manipulations undertaken to investigate gene function. An important boost to knowledge occurred in 2009 when, for the first time, the whole genome sequence of a Brachyspira strain (Brachyspira hyodysenteriae strain WA1) was obtained. The genomics analysis provided a significant increase in knowledge: for example, a previously unknown ~36 Kb plasmid was discovered and metabolic pathways were constructed. The study also revealed likely acquisition of genes involved in transport and central metabolic functions from other enteric bacterial species. Four subsequent publications have provided a similarly detailed analysis of other Brachyspira genomes, but of these only two included more than one strain of a species (20 strains of B. hyodysenteriae in one and three strains of B. pilosicoli in the other). Since then, more Brachyspira genomes have been made publicly available, with the sequences of at least one representative of each of the nine officially recognised species deposited at public genome repositories. All species have a single circular chromosome varying in size from ~2.5 to 3.3 Mb, with a C + G content of around 27%. In this chapter, we summarise the current knowledge and present a preliminary comparative genomic analysis conducted on 56 strains covering the official Brachyspira species. Besides providing detailed genetic maps of the bacteria, this analysis has revealed gene island rearrangements, putative phenotypes (including antimicrobial drug resistance) and genetic mutation mechanisms that enable brachyspires to evolve and respond to stress. The application of Next-Generation Sequencing (NGS) to generate genomic data from many more Brachyspira species and strains increasing will improve our understanding of these enigmatic spirochetes.

A longitudinal survey was conducted to investigate the presence of Brachyspira species in layer flocks. A total of 66 layer flocks kept in alternative husbandry systems were sampled at three time points: end of rearing, at peak of lay and at end of lay. Content from caecal samples of freshly killed birds was cultured at each sampling time point and processed for further investigations. Gross pathological lesions in caeca were recorded during post mortem investigation. Spirochaetes were isolated from 50 flocks: three flocks were positive at all three sampling points, 25 flocks at two and 22 flocks at one sampling point, respectively. The presence of Brachyspira spp. could not be related to specific gross pathological caecal lesions or antibiotic treatments. The number of positive flocks increased with the age of birds. Furthermore, organic flocks were more often positive than flocks from barn systems. In total 80 spirochaetal cultures were obtained. B. intermedia (43.8%) was the most common species, followed by B. pulli (13.8%) and B. pilosicoli (12.5%). Brachyspira murdochii and B. innocens were found in 5.0% and 2.5%, respectively, whereas 11.3% of Brachyspira isolates could not be identified to species level. In 11.3% of the samples mixed infections were detected. Finally, the longitudinal survey revealed for the first time a possible shift in the Brachyspira species in a substantial number (32%) of layer flocks during their lifetime.

During six years as German National Consultant Laboratory for Spirochetes we investigated 149 intestinal biopsies from 91 patients, which were histopathologically diagnosed with human intestinal spirochetosis (HIS), using fluorescence in situ hybridization (FISH) combined with 16S rRNA gene PCR and sequencing. Aim of this study was to complement histopathological findings with FISH and PCR for definite diagnosis and species identification of the causative pathogens. HIS is characterized by colonization of the colonic mucosa of the human distal intestinal tract by Brachyspira spp. Microbiological diagnosis of HIS is not performed, because of the fastidious nature and slow growth of Brachyspira spp. in culture. In clinical practice, diagnosis of HIS relies solely on histopathology without differentiation of the spirochetes. We used a previously described FISH probe to detect and identify Brachyspira spp. in histological gut biopsies. FISH allowed rapid visualization and identification of Brachyspira spp. in 77 patients. In most cases, the bright FISH signal already allowed rapid localization of Brachyspira spp. at 400× magnification. By sequencing, 53 cases could be assigned to the B. aalborgi lineage including "B. ibaraki" and "B. hominis", and 23 cases to B. pilosicoli. One case showed mixed colonization. The cases reported here reaffirm all major HIS Brachyspira spp. clusters already described. However, the phylogenetic diversity seems to be even greater than previously reported. In 14 cases, we could not confirm HIS by either FISH or PCR, but found colonization of the epithelium by rods and cocci, indicating misdiagnosis by histopathology. FISH in combination with molecular identification by 16S rRNA gene sequencing has proved to be a valuable addition to histopathology. It provides definite diagnosis of HIS and allows insights into phylogeny and distribution of Brachyspira spp. HIS should be considered as a differential diagnosis in diarrhea of unknown origin, particularly in patients from risk groups (e.g. patients with colonic adenomas, inflammatory polyps, inflammatory bowel disease or HIV infection and in men who have sex with men)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Swine dysentery (SD) is a mucohemorrhagic colitis of swine classically caused by infection with the intestinal spirochete Brachyspira hyodysenteriae Since around 2007, cases of SD have occurred in North America associated with a different strongly beta-hemolytic spirochete that has been molecularly and phenotypically characterized and provisionally named "Brachyspira hampsonii." Despite increasing international interest, B. hampsonii is currently not recognized as a valid species. To support its recognition, we sequenced the genomes of strains NSH-16 T , NSH-24, and P280/1, representing B. hampsonii genetic groups I, II, and III, respectively, and compared them with genomes of other valid Brachyspira species. The draft genome of strain NSH-16 T has a DNA G+C content of 27.4% and an approximate size of 3.2 Mb. Genomic indices, including digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and average amino acid identity (AAI), clearly differentiated B. hampsonii from other recognized Brachyspira species. Although discriminated genotypically, the three genetic groups are phenotypically similar. By electron microscopy, cells of different strains of B. hampsonii measure 5 to 10 μm by 0.28 to 0.34 μm, with one or two flat curves, and have 10 to 14 periplasmic flagella inserted at each cell end. Using a comprehensive evaluation of genotypic (gene comparisons and multilocus sequence typing and analysis), genomic (dDDH, ANI, and AAI) and phenotypic (hemolysis, biochemical profiles, protein spectra, antibiogram, and pathogenicity) properties, we classify Brachyspira hampsonii sp. nov. as a unique species with genetically diverse yet phenotypically similar genomovars (I, II, and III). We designate the type strain NSH-16 (= ATCC BAA-2463 = NCTC 13792)., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

An emerging novel spirochete in swine, provisionally designated "Brachyspira hampsonii," has been detected worldwide. It has been associated with swine dysentery and cannot be differentiated from B. hyodysenteriae, the classical etiologic agent of this disease, using standard phenotypic methods. We evaluated cross-reactions of "B. hampsonii" isolates recovered from avian species in some of the currently available species-specific polymerase chain reaction (PCR) assays for the identification of swine Brachyspira species. Ten avian "B. hampsonii" isolates recovered from wild waterfowl were used. No false-positive results were recorded with a B. pilosicoli-specific PCR based on the amplification of a fragment of the 16S rRNA gene. However, the percentage of false-positive results varied, with a range of 10-80%, in the evaluated B. hyodysenteriae-specific assays based on the amplification of the 23S rRNA, nox, and tlyA genes. Similarly, results of the B. intermedia-specific PCR assays yielded poor specificity, with up to 80% of the "B. hampsonii" isolates tested giving false-positive results. Finally, 2 "B. hampsonii" avian isolates yielded a positive result in a B. innocens- and B. murdochii-specific PCR. This result should be interpreted very cautiously as these 2 isolates could represent a recombinant genotype., (© 2016 The Author(s).)

The anaerobic spirochaete Brachyspira pilosicoli colonizes the large intestine of birds and mammals, including human beings, and may induce colitis and diarrhoea. B. pilosicoli has a recombinant population structure, and strains show extensive genomic rearrangements and different genome sizes. The resident chromosomal gene blaOXA-63 in B. pilosicoli encodes OXA-63, a narrow-spectrum group IV class D β-lactamase. Genes encoding four OXA-63 variants have been described in B. pilosicoli, and the current study was designed to investigate the distribution and diversity of such genes and proteins in strains of B. pilosicoli. PCRs were used to amplify blaOXA-63 group genes from 118 B. pilosicoli strains from different host species and geographical origins. One primer set was targeted externally to the gene and two sets were designed to amplify internal components. A total of 16 strains (13.6%) showed no evidence of possessing blaOXA-63 group genes, 44 (37.3%) had a full gene, 27 (22.9%) apparently had a gene but it failed to amplify with external primers, and 29 (24.6%) had only one or other of the two internal components amplified. Based on translation of the nucleotide sequences, ten new variants of the β-lactamase, designated OXA-470 through OXA-479, were identified amongst the 44 strains that had the full gene amplified. The 16 strains lacking blaOXA-63 group genes had a region of 1674 bp missing around where the gene was expected to reside. Despite apparent genomic rearrangements occurring in B. pilosicoli, positive selection pressures for conservation of blaOXA-63 group genes and OXA proteins appear to have been exerted.

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).

Outbreaks of bloody diarrhea in swine herds in the late 2000s signaled the reemergence of an economically significant disease, swine dysentery, in the United States. Investigations confirmed the emergence of a novel spirochete in swine, provisionally designated "Brachyspira hampsonii," with two genetically distinct clades. Although it has since been detected in swine and migratory birds in Europe and North America, little is known about its genetic diversity or its relationships with other Brachyspira species. This study characterizes B. hampsonii using a newly developed multilocus sequence typing (MLST) approach and elucidates the diversity, distribution, population structure, and genetic relationships of this pathogen from diverse epidemiological sources globally. Genetic characterization of 81 B. hampsonii isolates, originating from six countries, with our newly established MLST scheme identified a total of 20 sequence types (STs) belonging to three clonal complexes (CCs). B. hampsonii showed a heterogeneous population structure with evidence of microevolution locally in swine production systems, while its clustering patterns showed associations with its epidemiological origins (country, swine production system, and host species). The close genetic relatedness of B. hampsonii isolates from different countries and host species highlights the importance of strict biosecurity control measures. A comparative analysis of 430 isolates representing seven Brachyspira species (pathogens and commensals) from 19 countries and 10 host species depicted clustering by microbial species. It revealed the close genetic relatedness of B. hampsonii with commensal Brachyspira species and also provided support for the two clades of B. hampsonii to be considered a single species., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The Brachyspira species traditionally associated with swine dysentery and other diarrheal diseases in pigs are Brachyspira hyodysenteriae, Brachyspira pilosicoli, and, to a lesser extent, Brachyspira murdochii. "Brachyspira hampsonii" is a recently proposed novel species that causes clinical disease similar to that caused by B. hyodysenteriae. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems are increasingly available in veterinary diagnostic laboratories, are less expensive, and are faster than traditional microbiological and molecular methods for identification. Thirty-three isolates associated with Brachyspira species of importance to swine were added to an existing MALDI-TOF MS database library. In total, species included in the library were: B. hyodysenteriae, "B. hampsonii" clades I and II, Brachyspira innocens, Brachyspira intermedia, B. murdochii, and B. pilosicoli. A comparison between MALDI-TOF MS and nox sequencing was completed on 176 field isolates. Of the 176 field isolates, 174 (98.9%) matched species identification by both methods. Thirty field isolates were identified by both methods as "B. hampsonii". Twenty-seven of the 30 (90%) "B. hampsonii" field isolates matched clade designation in both assays. The nox sequencing identified 26 as "B. hampsonii" clade I and 4 as clade II. Comparatively, MALDI-TOF MS identified 25 of the 30 as "B. hampsonii" clade I and 5 as clade II. The current study indicates MALDI-TOF MS is a reliable tool for the identification of swine Brachyspira species; however, final clade designation for "B. hampsonii" may still require molecular techniques., (© 2014 The Author(s).)

Background: "Brachyspira hampsonii", discovered in North America in 2010 associated with dysentery-like illness, is an economically relevant swine pathogen resulting in decreased feed efficiency and increased morbidity, mortality and medication usage. "B. hampsonii" clade II strain 30446 has been shown to be causally associated with mucohemorrhagic diarrhea and colitis. Our objectives were to determine if "Brachyspira hampsonii" clade I strain 30599 is pathogenic to pigs, and to evaluate the relative diagnostic performance of three ante mortem sampling methodologies (direct PCR on feces, PCR on rectal GenoTube Livestock swabs, Brachyspira culture from rectal swabs). Five-week old pigs were intragastrically inoculated thrice with 108 genomic equivalents "B. hampsonii" (n = 12), or served as sham controls (n = 6). Feces were sampled and consistency assessed daily. Necropsies were performed 24 h after peak clinical signs., Results: One pig died due to unrelated illness. Nine of 11 inoculated pigs, but no controls, developed mucoid or mucohemorrhagic diarrhea (MHD). Characteristic lesions of swine dysentery were observed in large intestine. "B. hampsonii" strain 30599 DNA was detected by qPCR in feces of all inoculated pigs for up to 6 days prior to the onset of MHD. The organism was isolated from the feces and colons of pigs demonstrating MHD, but not from controls. B. intermedia was isolated from inoculated pigs without MHD, and from 5 of 6 controls., Conclusions: We conclude that "Brachyspira hampsonii" clade I strain 30599 is pathogenic and causes mucohemorrhagic diarrhea and colitis in susceptible pigs. Moreover, the three sampling methodologies performed similarly. GenoTube Livestock, a forensic swab designed to preserve DNA during shipping is a useful tool especially in settings where timely transport of diagnostic samples is challenging.

Unlabelled: The aim of this study consisted in evaluating MALDI-TOF MS as a tool for the identification of the genus Brachyspira (B.) and its relevant species for the pig industry. First, a database was created with 30 control strains, and superspectra for five different porcine Brachyspira species were calculated. In a second step, 67 field isolates were investigated using MALDI-TOF MS, and results were compared to those obtained using nox gene-based RFLP (reference method) and biochemical tests. Among the 67 field isolates, five different Brachyspira species were detected using nox gene-based RFLP analysis. MALDI-TOF MS analysis correctly assigned all isolates to the genus Brachyspira and identified all isolates from B. hyodysenteriae (29/29), B. pilosicoli (11/11), B. intermedia (4/4) and B. innocens (11/11). In terms of B. murdochii, MALDI-TOF MS assigned one of 12 isolates ambiguously as B. innocens/B. murdochii. The results of this study indicate that MALDI-TOF MS facilitates the diagnosis of swine dysentery and porcine intestinal spirochaetosis., Significance and Impact of the Study: Current methods for the discrimination of pathogenic Brachyspira hyodysenteriae and Brachyspira pilosicoli from Brachyspira species with low pathogenic potential have proven to be laborious and time-consuming and are therefore not suitable for routine diagnostics. This study describes the evaluation of MALDI-TOF MS for the identification of different porcine Brachyspira species in routine diagnostic laboratories. The results suggest that MALDI-TOF MS is an effective method for the identification of porcine Brachyspira spp. and accelerates diagnosis of swine dysentery and porcine intestinal spirochaetosis., (© 2013 The Society for Applied Microbiology.)

This report describes the detection of "Brachyspira (B.) hampsonii" clade I in Belgian pigs imported to Germany. Two of seventeen pigs from one herd were reported positive for Brachyspira hyodysenteriae by culture in a Belgian diagnostic laboratory, but negative for this Brachyspira species by specific PCR. In this study, from 22 fecal samples and 2 colon contents of these animals various Brachyspira species were cultured and identified by nox-RFLP as Brachyspira murdochii, Brachyspira innocens and Brachyspira intermedia. Albeit the six B. intermedia isolates proved to be negative in a species specific PCR. Sequencing of the nox-gene of three of these isolates revealed that the sequences were 99% identical to published sequences of "B. hampsonii" clade I. From one pig which was positive for "B. hampsonii" clade I histopathology was done and showed moderate lesions consistent with brachyspiral disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Anseriformes deserve special attention in the epidemiology of Brachyspira spp. because diverse Anseriformes species have been described to act as highly efficient carriers of several Brachyspira spp. that can also infect livestock. The aim of this study was to investigate the prevalence and diversity of Brachyspira spp. in waterfowl that winter in Spain. Brachyspira spp. were isolated from 51 of the 205 faecal samples collected from graylag geese and mallards in the Villafáfila Lagoons Nature Reserve (Northwestern Spain). The Brachyspira species identified through phenotyping, PCR and sequencing of the nox gene were B. pilosicoli (5.9%), B. alvinipulli (11.8%), "B. hampsonii" (19.6%), B. murdochii (23.5%) and B. innocens (39.2%). The most relevant finding of this study is the description of "B. hampsonii" in specimens from birds for the first time. Phylogenetic analysis of the nox gene sequences grouped all of the obtained "B. hampsonii" isolates into a cluster with Brachyspira strains previously identified by others as "B. hampsonii" and separated from other Brachyspira spp. isolates and reference strains. Additionally, this cluster was related to clades that grouped B. murdochii and B. innocens isolates. The identification of "B. hampsonii" was also achieved in 8 of the 10 isolates by sequencing the16S rRNA gene and tlyA gene. Regardless of the species identified, no antimicrobial resistance was observed in any of the enteropathogenic isolates recovered. This is the first description of "B. hampsonii" in European waterfowl, which might represent hosts that serve as natural reservoirs of this Brachyspira species. This finding indicates that this spirochete is not limited to North America, and its presence in wild birds in Europe poses a risk of transmission to livestock.

Brachyspira is associated with diarrhea and colitis in pigs, and control of these pathogens is complicated by their complex ecology. Identification of wildlife reservoirs of Brachyspira requires the discrimination of colonized animals and those simply contaminated through environmental exposure. Lesser snow geese (Chen caerulescens caerulescens) were sampled in the Canadian arctic during the summer of 2011, and cloacal swabs were cultured on selective media. Brachyspira isolates were obtained from 15/170 (8.8 %) samples, and 12/15 isolates were similar to isolates previously recovered from pigs, including "Brachyspira hampsonii", a recently characterized species associated with dysentery-like disease in pigs in North America. A pilot inoculation study with one strongly β-hemolytic B. hampsonii isolate resulted in fecal shedding of the isolate by inoculated pigs for up to 14 days post-inoculation, but no severe clinical disease. Results of this study indicate that lesser snow geese can be colonized by Brachyspira strains that can also colonize pigs. Millions of lesser snow geese (C. caerulescens caerulescens) travel through the major pork-producing areas of Canada and the USA during their annual migration, making them a potential factor in the continental distribution of these bacteria.

Brachyspira pilosicoli is an anaerobic intestinal spirochaete that colonizes the large intestine of various host species, in which it may induce diarrhoea, poor growth rates and a localized colitis known as intestinal (or colonic) spirochaetosis. The spirochaete is considered to be potentially zoonotic. The purpose of the current study was to develop a multiple-locus variable number tandem repeat analysis (MLVA) method as a simple and rapid tool to investigate the molecular epidemiology of B. pilosicoli. The genomic sequence of B. pilosicoli strain 95/1000 was analyzed for potential tandem repeats using the default parameters of the Tandem Repeat Finder program. A total of 22 repeat loci were identified and tested for their presence and variability on a set of 10 B. pilosicoli isolates. Five loci that were present in most isolates and that showed evidence of allelic variation were selected and used with a collection of 119 isolates from different host species and geographical locations. Not all the isolates amplified at all loci, but using the available data a total of 103 VNTR profiles were generated. The discriminatory power of this method was 0.976. A phylogenetic tree constructed from the allelic profiles confirmed the diversity of B. pilosicoli, and the general lack of clustering of strains based on species of origin or geographic origin. Some isolates with known epidemiological links were found to be identical or highly similar. The MLVA method was simple and easy to use, and could readily differentiate between strains of B. pilosicoli. MLVA should prove to be a useful tool for rapid identification of relationships between B. pilosicoli isolates in epidemiological investigations., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Swine dysentery is classically associated with infection by the strongly beta-hemolytic Brachyspira hyodysenteriae; however, the proposed novel species "Brachyspira hampsonii" has also been isolated from clinical cases of dysentery in the United States and Canada. Microbial culture is highly sensitive for detecting Brachyspira in clinical samples but requires several days for completion and is often followed by molecular testing for speciation. Alternatively, in situ hybridization using molecular probes applied to sections of formalin-fixed tissue can provide rapid, culture-independent identification of agents observed histologically. Accordingly, a fluorescent in situ hybridization assay was developed for confirmation of a clinical diagnosis of swine dysentery associated with infection by "B. hampsonii." An oligonucleotide probe (Hamp1210) targeting a specific 23S ribosomal RNA sequence of "B. hampsonii" was developed following sequence analysis and comparison of numerous Brachyspira spp. clinical isolates with reference sequences available in GenBank. The application of Hamp1210 and a previously published probe for B. hyodysenteriae (Hyo1210) to diseased colonic tissues successfully detected the target species in both experimentally infected pigs and naturally infected pigs from field cases, and the Hamp1210 probe consistently detected both clade I and clade II isolates of "B. hampsonii"; however, a strong positive signal was also observed in a single case where the Hamp1210 probe was applied to tissues infected with Brachyspira intermedia. In situ hybridization incorporating the Hamp1210 probe can reduce the delay from sample submission to pathogen identification in cases of swine dysentery associated with "B. hampsonii" infection where formalin-fixed tissues are available.

Spirochaetes belonging to the genus Brachyspira are anaerobic bacteria that colonize the large intestine of humans and animals, mainly pigs. The main species are namely, B. hyodysenteriae, the etiological agent of swine dysentery, B. pilosicoli, a zoonotic agent causing colonic spirochaetosis both in humans and different animal species, B. aalborgi, exclusively infecting humans causing colonic spirochaetosis, B. intermedia, a potential animal pathogen, B. innocens and B. murdochii, generally commensal of pigs, and B. alvinipulli, found in egg laying hens with diarrhea. In this study, for the first time, MALDI-TOF MS was applied on Brachyspira strains of human and animal origins, supplementing the existing database, limited to the species B. murdochii only, with spirochaetal protein profiles and demonstrating its usefulness in the rapid, cheap and reliable identification of Brachyspira strains at the species level, overcoming the problems previously encountered in the identification of these spirochaetes when using biochemical and genetic-based methods. Moreover, a dendrogram based on protein profiles of the different spirochaetal species was generated reflecting their host spectrum, showing in the same branch the only two species able to infect humans (B. aalborgi and B. pilosicoli) and in the other branch the spirochaetes infecting exclusively animals., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Multiple Brachyspira spp. can colonize the porcine colon, and the presence of the strongly beta-hemolytic Brachyspira hyodysenteriae is typically associated with clinical swine dysentery. Recently, several Brachyspira spp. have been isolated from the feces of pigs with clinical disease suggestive of swine dysentery, yet these isolates were not identified as B. hyodysenteriae by genotypic or phenotypic methods. This study used a mouse model of swine dysentery to compare the pathogenic potential of seventeen different Brachyspira isolates including eight atypical clinical isolates, six typical clinical isolates, the standard strain of B. hyodysenteriae (B204), and reference strains of Brachyspira intermedia and Brachyspira innocens. Results revealed that strongly beta-hemolytic isolates induced significantly greater cecal inflammation than weakly beta-hemolytic isolates regardless of the genetic identification of the isolate, and that strongly beta-hemolytic isolates identified as 'Brachyspira sp. SASK30446' and B. intermedia by PCR produced lesions indistinguishable from those caused by B. hyodysenteriae in this model., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Brachyspira spp. are frequent inhabitants of the chicken's intestine and some have been associated with enteric disease in humans. We studied contamination with Brachyspira spp. of carcasses of spent laying hens as a possible source of infections for humans and animals that may eat this meat. Eleven batches of hen carcasses, for a total of 110 carcasses, were bought in Belgian supermarkets during 2009-2010. Carcass rinse samples were examined for the presence of Brachyspira. Brachyspira spp. were cultured from some carcass in all batches. Besides presumably non-pathogenic species such as Brachyspira murdochii and Brachyspira innocens, the poultry pathogen Brachyspira intermedia and the poultry and suspected human pathogen Brachyspira pilosicoli were identified in 7/11 and 1/11 carcass batches, respectively, at high numbers, as shown using quantitative polymerase chain reactions. Multilocus sequence typing (MLST) demonstrated the presence of 2 and 13 MLST types of B. pilosicoli and B. intermedia, respectively, with all strains belonging to novel MLST types. The findings show that carcasses of spent laying hens are commonly contaminated with high numbers of Brachyspira spp., including the suspected zoonotic agent B. pilosicoli., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Classical swine dysentery is associated with the presence of the strongly beta-hemolytic Brachyspira hyodysenteriae. However, multiple Brachyspira spp. can colonize the porcine colon. Since 2008, several Brachyspira spp. not identified as B. hyodysenteriae by genotypic and/or phenotypic methods have been isolated from the feces of pigs with clinical disease typical of swine dysentery. In the current study, 8 clinical isolates, including 5 strongly beta-hemolytic and 3 weakly beta-hemolytic Brachyspira strains, and a reference strain of B. hyodysenteriae (B204) were inoculated into pigs (n = 6 per isolate) to compare pathogenic potential following oral inoculation. Results revealed that strongly beta-hemolytic isolates induced significantly greater typhlocolitis than those that are weakly beta-hemolytic, regardless of the genetic identification of the isolate, and that strongly beta-hemolytic isolates identified as "Brachyspira sp. SASK30446" and Brachyspira intermedia by polymerase chain reaction (PCR) produced lesions similar to those caused by B. hyodysenteriae. The results suggest that phenotypic culture characteristics of Brachyspira spp. may be a more sensitive indicator of potential to induce dysentery-like disease in pigs than molecular identification alone based on currently available PCR assays. Additionally, culture of mucosal scrapings obtained at necropsy was more sensitive than direct PCR on the same samples for detection of Brachyspira spp.

Since 2007, outbreaks of severe bloody diarrhea and hemorrhagic colitis have been reported in the United States and Canada. Though the primary causative agent of swine dysentery is Brachyspira hyodysenteriae, which is strongly hemolytic, the current report describes the isolation of a novel strongly hemolytic Brachyspira sp. This novel Brachyspira sp. was identified from clinical submissions at the Minnesota Veterinary Diagnostic Laboratory, and 40 of such isolates were obtained from 22 clinical submissions representing 5 states. Isolates were confirmed to be different from any known Brachyspira sp. on the basis of phylogenetic analysis of nucleotide sequences of nox and 16S ribosomal RNA (rRNA) genes. Phylogenetic analyses grouped all isolates into 2 clades (clades I and II), and grouping patterns were similar for both nox and 16S rRNA gene sequence analyses. Phenotypically, all isolates were indole and hippurate negative, and enzymatic profiling indicated 2 types of profiles, irrespective of the phylogenetic grouping, differing only in the production of β-glucosidase. The results suggest that a potentially virulent new species of Brachyspira sp., provisionally named "Brachyspira hampsonii ", is circulating among swine herds in the United States.

Different Brachyspira (B.) species colonize the porcine intestinal tract, some of which are pathogens of significant clinical and economic importance. In 2002 we published a novel molecular method for differentiation of Brachyspira species from pigs based on the amplification of the nox-gene and the generation of species-specific restriction patterns (nox-RFLP) using the enzymes BfmI and DpnII (Rohde et al., 2002). We applied this method for identification in addition to biochemical testing in doubtful cases until 2008. Since 2009 we have used it as the first line method of identification. The current study documents the results of examining 2050 Brachyspira isolates collected from January 2009 to December 2011. In addition to identifying isolates with previously described patterns, four novel restriction fragment length patterns were observed, and isolates with these patterns could be assigned to the species B. intermedia and the B. innocens/murdochii complex on the basis of their phenotypic properties and by nox-sequence analysis. In 2007 a potentially new Brachyspira species, "B. suanatina", was described in Swedish pigs (Råsbäck et al., 2007). From the published nox-gene sequence it could be expected that this Brachypira species should show a new restriction pattern making nox-RFLP a suitable technique for identification of "B. suanatina". In this study the new restriction fragment length pattern could be demonstrated in one of the strains described by Råsbäck et al. (AN4859/03). Nevertheless, no isolates with this new pattern corresponding to "B. suanatina" were identified amongst the 2050 Brachyspira isolates examined from northern Germany., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The distribution of many genes encoding virulence and virulence life-style (VL-S) factors in Brachyspira (B.) hyodysenteriae and other Brachyspira species are largely unknown. Their knowledge is essential e.g. for the improvement of diagnostic methods targeting the detection and differentiation of the species. Thus 121 German Brachyspira field isolates from diarrhoeic pigs were characterized down to the species level by restriction fragment length polymorphism analysis of the nox gene and subsequently subjected to polymerase chain reaction detecting VL-S genes for inner (clpX) and outer membrane proteins (OMPs: bhlp16, bhlp17.6, bhlp29.7, bhmp39f, bhmp39h), hemolysins (hlyA/ACP, tlyA), iron metabolism (ftnA, bitC), and aerotolerance (nox). For comparison, B. hyodysenteriae reference strains from the USA (n=7) and Australia (2) were used. Of all genes tested only nox was detected in all isolates. The simultaneous presence of both the tlyA and hlyA/ACP was restricted to the species B. hyodysenteriae. The hlyA infrequently occurred also in weakly hemolytic Brachyspira. Similarly to tlyA and hlyA all B. hyodysenteriae strains contained the ferritin gene ftnA which was also found in two Brachyspira intermedia isolates. OMP encoding genes were present in B. hyodysenteriae field isolates in rates of 0% (bhlp17.6, bhmp39h), 58.1% (bhlp29.7), and 97.3% (bhmp39f). Since the study revealed a high genetic heterogeneity among German B. hyodysenteriae field isolates differentiating them from USA as well as Australian strains, targets for diagnostic PCR were limited to the nox gene (genus specific PCR) as well as to the species specific nox(hyo) gene and the combination of hlyA and tlyA which allow to specifically detect B. hyodysenteriae., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Brachyspira species are fastidious anaerobic microorganisms, that infect the colon of various animals. The genus contains both important pathogens of livestock as well as commensals. Two species are known to infect humans: B. aalborgi and B. pilosicoli. There is some evidence suggesting that the veterinary pathogenic B. pilosicoli is a potential zoonotic agent, however, since diagnosis in humans is based on histopathology of colon biopsies, species identification is not routinely performed in human materials., Methods: The study population comprised 57 patients with microscopic evidence of Brachyspira infection and 26 patients with no histopathological evidence of Brachyspira infection. Concomitant faecal samples were available from three infected patients. Based on publically available 16S rDNA gene sequences of all Brachyspira species, species-specific primer sets were designed. DNA was extracted and tested by real-time PCR and 16S rDNA was sequenced., Results: Sensitivity and specificity for identification of Brachyspira species in colon biopsies was 100% and 87.7% respectively. Sequencing revealed B. pilosicoli in 15.4% of patients, B. aalborgi in 76.9% and a third species, tentatively named "Brachyspira hominis", in 26.2%. Ten patients (12.3%) had a double and two (3.1%) a triple infection. The presence of Brachyspira pilosicoli was significantly associated with inflammatory changes in the colon-biopsy (p=0.028)., Conclusions: This newly designed PCR allows for sub-differentiation of Brachyspira species in patient material and thus allows large-scaled surveillance studies to elucidate the pathogenicity of human Brachyspira infections. One-third of affected patients appeared to be infected with a novel species.

Brachyspira spp. are anaerobic intestinal spirochaetes that colonize vertebrates. Some species cause enteric diseases in pigs, chickens and possibly in humans, whereas others display a commensual relationship with their hosts. The aims were to investigate the prevalence among colonized free-living wild mallards (Anas platyrhynchos) of three enteropathogenic Brachyspira spp., and to describe the biodiversity of Brachyspira spp. isolates. Isolates from 150 birds were screened by PCR for 3 pathogenic Brachyspira spp., and 35 isolates from 20 mallards, 4 pigs and 1 chicken were subjected to phenotypic tests, 9 diagnostic PCRs, sequencing of the 16S rRNA and NADH oxidase (nox) genes, phylogenetic analysis and nox gene restriction enzyme analysis in silico. Of the 150 birds, 47%, 33% and 11% were positive by PCR for Brachyspira pilosicoli, Brachyspira intermedia and Brachyspira hyodysenteriae, respectively. Thirty-one characterized isolates were provisionally identified as B. intermedia, Brachyspira alvinipulli, "Brachyspira pulli", or B. pilosicoli, whereas 4 were of indeterminate species affiliation. Many isolates were phylogenetically related to isolates from livestock. Isolates identified by PCR as B. pilosicoli displayed particularly high biodiversity. Up to five different Brachyspira genotypes were found from the same bird. Sequencing of amplicons from isolates that displayed ambiguous results as judged from PCR and phenotyping showed that several diagnostic PCRs were non-specific. Nox gene restriction enzyme analysis in silico correctly identified 2 of 34 characterized isolates. A culture technique based on filtration that produced uncontaminated spirochaete isolates was described. The results show that mallard intestines support a high degree of biodiversity among Brachyspira spp., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The aim of the current study was to look for evidence of possible cross-species transmission of Brachyspira species between rodents and farm animals. To do this, previously collected and characterised Brachyspira isolates from rodents, pigs and chickens on the same farms were analysed by random amplified polymorphic DNA (RAPD). Isolates with similar RAPD banding patterns were further typed by pulsed-field gel electrophoresis (PFGE). Identical isolates of Brachyspira pilosicoli, Brachyspira intermedia, Brachyspira murdochii and Brachyspira innocens from pigs and rodents and of B. murdochii from laying hens and rodents were found, indicating cross-species transmission at farm level. PFGE data from rodent isolates of Brachyspira hyodysenteriae were compared with PFGE data from previously typed field isolates of B. hyodysenteriae from pigs with swine dysentery and isolates from mallards (Anas platyrhynchos). Three of four isolates of B. hyodysenteriae from rodents were similar to porcine field isolates by PFGE. PCR analyses of the plasmid-encoded and potential virulence determinants rfb genes B, A, D and C showed that they were present in isolates of B. hyodysenteriae of porcine, mallard and rodent origin., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Typhlocolitis and dysentery due to Brachyspira hyodysenteriae infection represent an economically important disease syndrome in growing pigs. Largely disappearing from U.S. swine herds in the late 1990 s and early 2000s, Brachyspira-associated disease and bacterial isolation from swine with clinical disease has increased in the last several years, and non-B. hyodysenteriae isolates are commonly identified. Antimicrobial resistance has been demonstrated in Brachyspira spp. isolates from Europe and Asia, and may be the reason for the resurgence in U.S. herds. Seventy-nine clinical isolates identified at the Iowa State University Veterinary Diagnostic Lab were tested with multiple polymerase chain reaction assays to establish species identity, and evaluated for minimum inhibitory concentrations (MICs) using an agar dilution method against lincomycin, gentamicin, valnemulin, tiamulin, salinomycin, and carbadox. Only 38.0% of isolates could be confirmed as the known pathogens B. hyodysenteriae (30.4%) or Brachyspira pilosicoli (7.6%). Twenty of the 79 isolates (25.3%) were identified as Brachyspira murdochii, and 13.9% could not be identified to species. The MIC values were consistently high against lincomycin and moderately high against gentamicin. The remaining antimicrobials had MICs that were at the low end of the test ranges. Brachyspira murdochii and Brachyspira spp. had significantly greater MIC values against several of these drugs than other Brachyspira spp. examined. The increased incidence of these less definitively characterized Brachyspira species with increased MIC values to commonly prescribed antimicrobials may, at least in part, explain the increased prevalence and severity of this disease complex in recent years. Further research is necessary to understand these changes.

Four quantitative PCR (qPCR) assays were evaluated for quantitative detection of Brachyspira pilosicoli, Lawsonia intracellularis, and E. coli fimbrial types F4 and F18 in pig feces. Standard curves were based on feces spiked with the respective reference strains. The detection limits from the spiking experiments were 10(2) bacteria/g feces for Bpilo-qPCR and Laws-qPCR, 10(3)CFU/g feces for F4-qPCR and F18-qPCR. The PCR efficiency for all four qPCR assays was between 0.91 and 1.01 with R(2) above 0.993. Standard curves, slopes and elevation, varied between assays and between measurements from pure DNA from reference strains and feces spiked with the respective strains. The linear ranges found for spiked fecal samples differed both from the linear ranges from pure culture of the reference strains and between the qPCR tests. The linear ranges were five log units for F4-qPCR, and Laws-qPCR, six log units for F18-qPCR and three log units for Bpilo-qPCR in spiked feces. When measured on pure DNA from the reference strains used in spiking experiments, the respective log ranges were: seven units for Bpilo-qPCR, Laws-qPCR and F18-qPCR and six log units for F4-qPCR. This shows the importance of using specific standard curves, where each pathogen is analysed in the same matrix as sample DNA. The qPCRs were compared to traditional bacteriological diagnostic methods and found to be more sensitive than cultivation for E. coli and B. pilosicoli. The qPCR assay for Lawsonia was also more sensitive than the earlier used method due to improvements in DNA extraction. In addition, as samples were not analysed for all four pathogen agents by traditional diagnostic methods, many samples were found positive for agents that were not expected on the basis of age and case history. The use of quantitative PCR tests for diagnosis of enteric diseases provides new possibilities for veterinary diagnostics. The parallel simultaneous analysis for several bacteria in multi-qPCR and the determination of the quantities of the infectious agents increases the information obtained from the samples and the chance for obtaining a relevant diagnosis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The aims of this study were to use multilocus sequence typing (MLST) to (i) investigate the population structure, diversity and molecular epidemiology of the weakly haemolytic anaerobic intestinal spirochaete Brachyspira intermedia, and (ii) determine the relationship of the species to the other two indole-positive but strongly haemolytic Brachyspira species--B. hyodysenteriae and "B. suanatina". Seventy-seven B. intermedia isolates from pigs and chickens were analysed, with the nucleotide sequences of seven conserved genomic loci examined for each. B. intermedia was genetically diverse, with the 77 isolates being divided into 71 sequence types (STs) and 64 amino acid types (AATs). Many distinct groups of B. intermedia isolates were identified, with some isolates being separated from others by large genetic distances. Although both pig and chicken isolates were found in most groups, suggesting that cross-species transmission of such isolates may occur, some isolates from pigs were located in small groups that did not include chicken isolates, and vice versa. Eight clonal complexes (Cc) of STs were identified by e-Burst analysis. The Ccs contained between 2 and 5 STs, and between 2 and 9 isolates. Five Ccs contained multiple isolates from the same farms, collected at the same time, indicating the existence of ongoing minor genetic change amongst isolates at the farm level. On the other hand, isolates with quite different STs also were found amongst multiple isolates collected from some farms. By comparison with the much more restricted diversity observed for 111 isolates of B. hyodysenteriae, and 4 isolates of "B. suanatina", it is difficult to justify including all weakly haemolytic indole-positive Brachyspira isolates in the single species B. intermedia., ((c) 2009 Elsevier B.V. All rights reserved.)

A histological diagnosis of human intestinal spirochaetosis (HIS) was made in 114 patients during the period 1994-2007. All patients lived in three prefectures in the northern part of Honshu, Japan. Most patients were elderly and male. Twenty-nine patients complained of abdominal pain, bloody stools, diarrhoea or bowel symptoms, but most patients showed no direct symptoms of bowel disease, and occult faecal blood detected at medical check-up was the main reason for colonoscopic examination. There were no homosexual patients and no immunosuppressed patients. HIS was evenly distributed throughout the whole colorectum. PCR analysis of Brachyspira aalborgi and Brachyspira pilosicoli revealed that more patients were infected with B. aalborgi. Follow-up PCR studies confirmed that infestation with B. aalborgi could be repeatedly detected over a 6 year period. This study, involving over 100 patients, identified the characteristic features of HIS in northern Japan. The results suggest that these spirochaetes may be harmless commensals that cause no obvious pathological alterations in infected individuals.

Anaerobic intestinal spirochetes (genus Brachyspira) include several species that are recognized as pathogens of poultry. Surveys undertaken in Europe and Australia have shown that layer and breeder flocks are often colonized by the pathogenic species Brachyspira intermedia and Brachyspira pilosicoli, but similar surveys have not been conducted in the United States. In the current study, DNA was extracted from fecal samples (n=50) collected from each of 21 flocks of laying hens >40 wk of age in Pennsylvania, and this material was tested for B. intermedia and B. pilosicoli using a duplex PCR. Negative samples also were tested using a Brachyspira genus-specific PCR. The consistency of the feces was observed, and manure handling systems and medication histories were recorded. Brachyspira intermedia was detected in 662 (63.1%) samples from 17 (81%) flocks, with a within-flock prevalence of 10%-100%. Brachyspira pilosicoli was detected in 112 (10.7%) samples from 5 flocks (23.8%), with a within-flock prevalence of 8%-82%. Four of the flocks had both pathogenic species present, three had no pathogenic species detected, and two had no Brachyspira species detected. Nine flocks had many fecal samples with a wet appearance and/or a caramel color, and all of these were colonized with one or the other of the two pathogenic species. Nine of 12 flocks with manure that was mainly dry also were colonized. Differences in colonization rates between flocks with or without wet manure were not significant. Colonization with pathogenic Brachyspira species, and particularly B. intermedia, occurs very commonly in layer flocks >40 wk of age in Pennsylvania. The significance of this high rate of colonization requires further investigation.

The microbes that accompany the etiologic agent of cholera, Vibrio cholerae, are only now being defined. In this study, spirochetes from the genus Brachyspira were identified at high titers in more than one third of cholera patients in Bangladesh. Spirochetosis should now be tracked in the setting of cholera outbreaks.

The prevalence of infections with different Brachyspira species was assessed in 202 pigs with various chronic herd problems using different methods. Twenty-seven pigs (13.4%) were positive for Brachyspira spp. with at least one of the methods used. The highest number of positives was identified with mucosal scraping-PCR (23), followed by PET-PCR (22) and bacteriological-biochemical analysis (15). With the exception of three cases of B. pilosicoli infections, only weakly pathogenic Brachyspira species were identified. The majority was B. murdochii, followed by B. innocens and B. intermedia. Concurrent infections with two or more Brachyspira species were common and accounted for 37.1% of the total. Presence of weakly haemolytic Brachyspira was associated with wasting and diarrhoea in a number of cases. This investigation shows that infections with weakly haemolytic Brachyspira spp. may contribute to colonic pathology in pigs with chronic herd problems and that mixed infections seem to occur more frequently than previously noticed.

Brachyspira pilosicoli is an anaerobic spirochaete that colonizes the large intestine of humans and various species of animals and birds. The spirochaete is an important enteric pathogen of pigs and poultry, but its pathogenic potential in humans is less clear. In the current study, the occurrence of B. pilosicoli in faecal samples from 766 individuals in two different population groups in Perth, Western Australia, was investigated by selective anaerobic culture. Of 586 individuals who were long-term residents of Perth, including children, elderly patients in care and in hospital and individuals with gastrointestinal disease, only one was culture positive. This person had a history of diverticulitis. In comparison, faeces from 17 of 180 (9.4 %) Indonesians who were short- or medium-term visitors to Perth were positive for B. pilosicoli. The culture-positive individuals had been in the city for between 10 days and 4.5 years (median 5 months). Resampling of subsets of the Indonesians indicated that all negative people remained negative and that some positive individuals remained positive after 5 months. Two individuals had pairs of isolates recovered after 4 and 5 months that had the same PFGE types, whilst another individual had isolates with two different PFGE types that were identified 2 months apart. Individuals who were culture-positive were likely to have been either colonized in Indonesia before arriving in Perth or infected in Perth following contact with other culture-positive Indonesians with whom they socialized. Colonization with B. pilosicoli was not significantly associated with clinical signs at the time the individuals were tested, although faeces with wet-clay consistency were 1.5 times more likely (confidence interval 0.55-4.6) than normal faeces to contain B. pilosicoli.

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).

PCR assays designed to amplify DNA from the anaerobic intestinal spirochaete Brachyspira aalborgi were conducted on DNA extracted from 938 faecal samples from 469 residents on the Indonesian island of Bali. The individuals tested were sampled twice in one year and were from four rural villages, one peri-urban centre and the capital city, Denpasar. Overall, an unexpectedly high prevalence of colonization (24.7%) was found, with prevalence rates at different locations varying from a low of 15.6% at one village to 41.5% in the peri-urban centre. Comparison of prevalence rates at the two sampling times suggested that, in many individuals, colonization was likely to be prolonged (>3 months) and/or that reinfection was occurring frequently in these people. Analysis of a questionnaire administered to the individuals who were sampled identified specific risk factors for colonization as location, co-colonization with the related intestinal spirochaete Brachyspira pilosicoli and use of drinking water obtained from wells rather than from taps. No specific associations with clinical symptoms were identified.

Several species of intestinal spirochaetes, Brachyspira (B.) alvinipulli, B. intermedia and B. pilosicoli, may cause reduced egg production and faecal staining of eggshells in chickens. The aim of this study was to characterize potentially pathogenic and presumably non-pathogenic Brachyspira spp. from commercial laying hens. Selective culture, phenotyping, PCR and 16S rRNA gene sequencing were used and clinical data were collected. Phenotypic profiles were obtained for 489 isolates and 351 isolates obtained after subculture, and 30 isolates were selected for molecular characterization. Seven isolates were positive by a B. intermedia-specific PCR based on the nox gene, and two were positive in a B. hyodysenteriae-specific 23S rRNA gene based PCR. By comparative phylogenetic analysis in combination with PCR and phenotyping, seven isolates were identified as B. intermedia, eight isolates as B. innocens, five as B. murdochii, and three isolates each as B. alvinipulli and "B. pulli". The remaining four isolates could not be assigned to any presently recognized species. Co-infection with several species or genetic variants of Brachyspira spp. were detected in some flocks and samples, suggesting a high level of diversity. Organic flocks with access to outdoor areas were at higher risk (RR=2.3; 95% CI 1.5-3.6) for being colonized than chickens in other housing systems. No significant differences between colonized and non-colonized flocks were found regarding clinical parameters, i.e. mortality, egg production, faecally contaminated eggshells, and wet litter. Our results show that a combination of traditional laboratory diagnostics, molecular tests and phylogeny is needed for identification of Brachyspira sp. from chickens.

We examined the usefulness of PCR-based restriction fragment length polymorphism (PCR-RFLP) and species-specific PCR combined with a newly devised rapid biochemical test using microplates for identifying weakly beta-hemolytic intestinal spirochetes (WBHIS) isolated from pigs. WBHIS strains showing atypical biochemical characteristics were decisively identified at the species level by PCR-RFLP and species-specific PCR. Identification of WBHIS at the species level in routine diagnostic work will certainly contribute to clarifying the pathogenicity of WBHIS.

Cecal samples from laying chickens from 25 farms with a history of decreased egg production, diarrhea, and/or increased feed conversion ratios were examined for anaerobic intestinal spirochetes of the genus Brachyspira. Seventy-three samples positive in an immunofluorescence assay for Brachyspira species were further examined using selective anaerobic culture, followed by phenotypic analysis, species-specific PCRs (for Brachyspira hyodysenteriae, B. intermedia, and B. pilosicoli), and a Brachyspira genus-specific PCR with sequencing of the partial 16S rRNA gene products. Brachyspira cultures were obtained from all samples. Less than half of the isolates could be identified to the species level on the basis of their biochemical phenotypes, while all but four isolates (5.2%) were speciated by using PCR and sequencing of DNA extracted from the bacteria. Different Brachyspira spp. were found within a single flock and also in cultures from single chickens, emphasizing the need to obtain multiple samples when investigating outbreaks of avian intestinal spirochetosis. The most commonly detected spirochetes were the pathogenic species B. intermedia and B. pilosicoli. The presumed nonpathogenic species B. innocens, B. murdochii, and the proposed "B. pulli" also were identified. Pathogenic B. alvinipulli was present in two flocks, and this is the first confirmed report of B. alvinipulli in chickens outside the United States. Brachyspira hyodysenteriae, the agent of swine dysentery, also was identified in samples from three flocks. This is the first confirmed report of natural infection of chickens with B. hyodysenteriae. Experimental infection studies are required to assess the pathogenic potential of these B. hyodysenteriae isolates.

Six Brachyspira type and reference strains, and 14 well characterized porcine field isolates representing all recognised porcine Brachyspira spp. were compared by different molecular methods. Sequence analysis of the 16S rRNA and the nox genes, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) were used in the study. In addition the isolates were analysed by five species-specific PCR systems. The topologies of the dendrograms obtained from each of the four typing systems were different. The B. pilosicoli isolates formed monophyletic clusters in all dendrograms, but with different sister lines. All five porcine Brachyspira species formed monophyletic clusters in the nox gene-based dendrogram only. All five PCR systems accurately identified their targets, except for the nox gene-based B. intermedia-specific system, by which it was not possible to identify one of the presumed B. intermedia isolates, and the other B. intermedia-specific system, based on the 23S rRNA gene, gave a positive reaction for one B. innocens isolate. In an extended study, 46 additional isolates and the original eight isolates with the phenotypes of B. hyodysenteriae or B. intermedia were compared by PFGE and PCR. The PFGE results indicated a high genetic diversity of isolates with the phenotype of B. intermedia. Thirty-three of 34 tested isolates could be identified by one or both of the two B. intermedia-specific PCR systems used, however, only 19 of the 34 isolates were positive in both systems.

The purpose of this study was to evaluate a multilocus sequence typing (MLST) scheme for intestinal spirochaetes of the genus Brachyspira. Eight loci mainly coding for enzymes previously used in multilocus enzyme electrophoresis analysis of Brachyspira species were examined in 66 Brachyspira field isolates and type/reference strains. The isolates and strains were recovered from pigs, birds, dogs and a mouse and originated from seven European countries, the USA and Canada. Forty-six isolates represented recognized Brachyspira species and 20 represented provisionally designated species or isolates that have not been classified. Only two loci gave PCR products for all 66 strains and isolates, but amplicons for seven loci were obtained for 44 of the isolates. Sequences for each locus had a DNA allelic variation of 30-47 and an amino acid allelic variation of 14-47 that gave rise to the same number of sequence and amino acid types (58) for the strains and isolates studied. A population snapshot based on sequence and amino acid types showed a close phylogenetic relationship amongst the porcine isolates from the same geographical regions, and indicated a close evolutionary relationship between isolates recovered from pigs and mallards. A general concordance was obtained between the MLST groupings and classifications based on culture and biochemical tests, 16S rDNA sequence analysis and random amplified polymorphic DNA analysis. This is a first step towards establishing an MLST system for use in identifying Brachyspira species and determining relationships between individual strains and species in the genus.