Brachyspira catarrhinii sp. nov., an anaerobic intestinal spirochaete isolated from vervet monkeys may have been misidentified as Brachyspira aalborgi in previous studies.

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 Z12T 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.

Weakly haemolytic variants of Brachyspira hyodysenteriae newly emerged in Europe belong to a distinct subclade with unique genetic properties.

Brachyspira (B.) hyodysenteriae is widespread globally, and can cause mucohaemorrhagic colitis (swine dysentery, SD) with severe economic impact in infected herds. Typical strains of B. hyodysenteriae are strongly haemolytic on blood agar, and the haemolytic activity is believed to contribute to virulence in vivo. However, recently there have been reports of atypical weakly haemolytic isolates of B. hyodysenteriae (whBh). In this study, 34 European whBh and 82 strongly haemolytic isolates were subjected to comparative genomic analysis. A phylogenetic tree constructed using core single nucleotide polymorphisms showed that the whBh formed a distinct sub-clade. All eight genes previously associated with haemolysis in B. hyodysenteriae were present in the whBh. No consistent patterns of amino acid substitutions for all whBh were found in these genes. In contrast, a genome region containing six coding sequences (CDSs) had consistent nucleotide sequence differences between strongly and whBh isolates. Two CDSs were predicted to encode ABC transporter proteins, and a TolC family protein, which may have a role in the export of haemolysins from B. hyodysenteriae. Another difference in this region was the presence of three CDSs in whBh that are pseudogenes in strongly haemolytic isolates. One of the intact CDSs from whBh encoded a predicted PadR-like transcriptional repressor that may play a role in repression of haemolysis functions. In summary, a sub-clade of whBh isolates has emerged in Europe, and several genomic differences, that potentially explain the weakly haemolytic phenotype, were identified. These markers may provide targets for discriminatory molecular tests needed in SD surveillance.

An Investigation into the Etiological Agents of Swine Dysentery in Australian Pig Herds.

Swine dysentery (SD) is a mucohemorrhagic colitis, classically seen in grower/finisher pigs and caused by infection with the anaerobic intestinal spirochete Brachyspira hyodysenteriae. More recently, however, the newly described species Brachyspira hampsonii and Brachyspira suanatina have been identified as causing SD in North America and/or Europe. Furthermore, there have been occasions where strains of B. hyodysenteriae have been recovered from healthy pigs, including in multiplier herds with high health status. This study investigated whether cases of SD in Australia may be caused by the newly described species; how isolates of B. hyodysenteriae recovered from healthy herds compared to isolates from herds with disease; and how contemporary isolates compare to those recovered in previous decades, including in their plasmid gene content and antimicrobial resistance profiles. In total 1103 fecal and colon samples from pigs in 97 Australian herds were collected and tested. Of the agents of SD only B. hyodysenteriae was found, being present in 34 (35.1%) of the herds, including in 14 of 24 (58%) herds that had been considered to be free of SD. Multilocus sequence typing applied to 96 isolates from 30 herds and to 53 Australian isolates dating from the 1980s through the early 2000s showed that they were diverse, distinct from those reported in other countries, and that the 2014/16 isolates generally were different from those from earlier decades. These findings provided evidence for ongoing evolution of B. hyodysenteriae strains in Australia. In seven of the 20 herds where multiple isolates were available, two to four different sequence types (STs) were identified. Isolates with the same STs also were found in some herds with epidemiological links. Analysis of a block of six plasmid virulence-associated genes showed a lack of consistency between their presence or absence and their origin from herds currently with or without disease; however, significantly fewer isolates from the 2000s and from 2014/16 had this block of genes compared to isolates from the 1980s and 1990s. It is speculated that loss of these genes may have been responsible for the occurrence of milder disease occurring in recent years. In addition, fewer isolates from 2014/16 were susceptible to the antimicrobials lincomycin, and to a lesser extent tiamulin, than those from earlier Australian studies. Four distinct multi-drug resistant strains were identified in five herds, posing a threat to disease control.

Characterization and Recognition of Brachyspira hampsonii sp. nov., a Novel Intestinal Spirochete That Is Pathogenic to Pigs.

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-16T, 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-16T 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).

Brachyspira hyodysenteriae isolated from apparently healthy pig herds following an evaluation of a prototype commercial serological ELISA.

Swine dysentery (SD) is a disease mainly of grower/finisher pigs characterised by severe mucohaemorrhagic colitis. The classical aetiological agent is the anaerobic intestinal spirochaete Brachyspira hyodysenteriae, although "Brachyspira hampsonii" and Brachyspira suanatina also cause SD. This study reports on the unexpected isolation of B. hyodysenteriae from pigs in apparently healthy herds that gave positive reactions when tested with a prototype commercial serological ELISA for detecting herds infected with B. hyodysenteriae (Priocheck(®)Brachyspira porcine Ab ELISA). The ELISA was tested with sera collected at abattoirs from 1770 slaughtered pigs from 30 Australian herds, including 12 with a history of SD and18 that were considered by their consulting veterinarians to be healthy. The latter herds had no history of SD and did not routinely use antimicrobials that may have masked the disease. Based on the recommended ELISA cut-off value, 25 herds were recorded as showing evidence of infection, including 11 of 12 herds that were considered infected by the submitters and 14 of the 18 "healthy" herds. When faecal or colonic wall samples from 11 of the 14 "false positive" herds subsequently were culturing 6-24 months after the original ELISA testing was completed, different strains of B. hyodysenteriae were isolated from six herds, including a high-health status breeding herd. The existence of apparently healthy herds that are colonised by B. hyodysenteriae has major implications for the control of SD. Had the ELISA not been trialled it is unlikely that colonic samples from these herds would have been cultured and the colonisation identified.

Development of a serological ELISA using a recombinant protein to identify pig herds infected with Brachyspira hyodysenteriae.

Brachyspira hyodysenteriae is an anaerobic spirochaete that can induce swine dysentery (SD), a severe mucohaemorrhagic colitis in grower and fattener pigs. The aim of this study was to develop a serological ELISA for use as a screening method to detect evidence of herd infection. Bioinformatic analysis of the complete genome sequence of strain WA1 was used to identify genes predicted to encode outer membrane proteins. Twenty candidate genes were expressed in an Escherichia coli mediated system, and purified as histidine-tagged recombinant proteins. Selection of optimal antigens under different conditions was conducted using Western blot and ELISA with a range of pig sera from infected and uninfected pigs. From this analysis, three recombinant proteins were selected as being most suitable for use as antigens. These antigens then were tested under optimized conditions in an indirect ELISA detecting IgG2 using 1551 sera from healthy pigs at slaughter, comprising 896 from 18 herds considered to be free from SD and 655 from 12 infected herds. Using a cut-off value for positivity of the mean plus five standard deviations of the mean for the negative sera, the best overall results were obtained with the ELISA using antigen H114, which was 100% specific and 91.7% sensitive at detecting the reported status of the herds. This new ELISA should be a useful adjunct for detecting and monitoring the status of herds with respect to the presence of B. hyodysenteriae, and should prove useful for understanding the dynamics of infection in herds where the spirochaete is present.

Genes encoding ten newly designated OXA-63 group class D ß-lactamases identified in strains of the pathogenic intestinal spirochaete Brachyspira pilosicoli.

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 ß-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 ß-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.

Emergence of Brachyspira species and strains: reinforcing the need for surveillance.

This short review discusses the increasing complexity that has developed around the understanding of Brachyspira species that infect pigs, and their ability to cause disease. It describes the recognition of new weakly haemolytic Brachyspira species, and the growing appreciation that Brachyspira pilosicoli and some other weakly haemolytic species may be pathogenic in pigs. It discusses swine dysentery (SD) caused by the strongly haemolytic Brachyspira hyodysenteriae, particularly the cyclical nature of the disease whereby it can largely disappear as a clinical problem from a farm or region, and re-emerge years later. The review then describes the recent emergence of two newly described strongly haemolytic pathogenic species, "Brachyspira suanatina" and "Brachyspira hampsonii" both of which appear to have reservoirs in migratory waterbirds, and which may be transmitted to and between pigs. "B. suanatina" seems to be confined to Scandinavia, whereas "B. hampsonii" has been reported in North America and Europe, causes a disease indistinguishable from SD, and has required the development of new routine diagnostic tests. Besides the emergence of new species, strains of known Brachyspira species have emerged that vary in important biological properties, including antimicrobial susceptibility and virulence. Strains can be tracked locally and at the national and international levels by identifying them using multilocus sequence typing (MLST) and comparing them against sequence data for strains in the PubMLST databases. Using MLST in conjunction with data on antimicrobial susceptibility can form the basis for surveillance programs to track the movement of resistant clones. In addition some strains of B. hyodysenteriae have low virulence potential, and some of these have been found to lack the B. hyodysenteriae 36 kB plasmid or certain genes on the plasmid whose activity may be associated with colonization. Lack of the plasmid or the genes can be identified using PCR testing, and this information can be added to the MLST and resistance data to undertake detailed surveillance. Strains of low virulence are particularly important where they occur in high health status breeding herds without causing obvious disease: potentially they could be transmitted to production herds where they may colonize more effectively and cause disease under stressful commercial conditions.

Absence of a set of plasmid-encoded genes is predictive of reduced pathogenic potential in Brachyspira hyodysenteriae.

The gene content of 14 strains of the intestinal spirochaete Brachyspira hyodysenteriae was compared using a DNA microarray. A consistent difference occurred in a block of four genes on the ~36 Kb plasmid, with these being present in six virulent strains and absent in eight strains with reduced pathogenic potential. These genes encoded a predicted radical S-adenosylmethionine domain protein, a glycosyl transferase group 1-like protein, an NAD dependent epimerase and a dTDP-4-dehydrorhamnose 2-5 epimerase: they may be involved in rhamnose biosynthesis and glycosylation. The absence of these plasmid genes in B. hyodysenteriae isolates is predictive of reduced pathogenic potential.

Intestinal spirochaetes (Brachyspira spp.) colonizing flocks of layer and breeder chickens in Malaysia.

Avian intestinal spirochaetosis causes problems including delayed onset of lay and wet litter in adult chickens, and results from colonization of the caecae/rectum with pathogenic intestinal spirochaetes (genus Brachyspira). Because avian intestinal spirochaetosis has not previously been studied in South East Asia, this investigation was undertaken in Malaysia. Faecal samples were collected from 25 farms and a questionnaire was administered. Brachyspira species were detected by polymerase chain reaction in 198 of 500 (39%) faecal samples from 20 (80%) farms, including 16 (94%) layer and four (50%) breeder farms. Pathogenic Brachyspira pilosicoli was identified in five (29%) layer and two (25%) breeder farms whilst pathogenic Brachyspira intermedia was detected in nine (53%) layer and one (12.5%) of the breeder farms. Twelve (80%) layer farms had egg production problems and 11 (92%) were positive for Brachyspira: three (25%) for B. pilosicoli and six (50%) for B. intermedia. Of three breeder farms with egg production problems, one was colonized with B. pilosicoli. Three of ten layer farms with wet litter were positive for B. pilosicoli and six for B. intermedia. Of four breeder farms with wet litter, one was colonized with B. pilosicoli and one with B. intermedia. No significant associations were found between colonization and reduced egg production or wet litter, perhaps because so many flocks were colonized. A significant association (P = 0.041) occurred between a high prevalence of colonization and faecal staining of eggs. There were significant positive associations between open-sided housing (P = 0.006), and flocks aged >40 weeks (P < 0.001) and colonization by pathogenic species.

Comparative genomics of Brachyspira pilosicoli strains: genome rearrangements, reductions and correlation of genetic compliment with phenotypic diversity.

BACKGROUND: The anaerobic spirochaete Brachyspira pilosicoli causes enteric disease in avian, porcine and human hosts, amongst others. To date, the only available genome sequence of B. pilosicoli is that of strain 95/1000, a porcine isolate. In the first intra-species genome comparison within the Brachyspira genus, we report the whole genome sequence of B. pilosicoli B2904, an avian isolate, the incomplete genome sequence of B. pilosicoli WesB, a human isolate, and the comparisons with B. pilosicoli 95/1000. We also draw on incomplete genome sequences from three other Brachyspira species. Finally we report the first application of the high-throughput Biolog phenotype screening tool on the B. pilosicoli strains for detailed comparisons between genotype and phenotype. RESULTS: Feature and sequence genome comparisons revealed a high degree of similarity between the three B. pilosicoli strains, although the genomes of B2904 and WesB were larger than that of 95/1000 (~2,765, 2.890 and 2.596 Mb, respectively). Genome rearrangements were observed which correlated largely with the positions of mobile genetic elements. Through comparison of the B2904 and WesB genomes with the 95/1000 genome, features that we propose are non-essential due to their absence from 95/1000 include a peptidase, glycine reductase complex components and transposases. Novel bacteriophages were detected in the newly-sequenced genomes, which appeared to have involvement in intra- and inter-species horizontal gene transfer. Phenotypic differences predicted from genome analysis, such as the lack of genes for glucuronate catabolism in 95/1000, were confirmed by phenotyping. CONCLUSIONS: The availability of multiple B. pilosicoli genome sequences has allowed us to demonstrate the substantial genomic variation that exists between these strains, and provides an insight into genetic events that are shaping the species. In addition, phenotype screening allowed determination of how genotypic differences translated to phenotype. Further application of such comparisons will improve understanding of the metabolic capabilities of Brachyspira species.

Dissemination of clonal groups of Brachyspira hyodysenteriae amongst pig farms in Spain, and their relationships to isolates from other countries.

BACKGROUND: Swine dysentery (SD) is a widespread diarrhoeal disease of pigs caused by infection of the large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Understanding the dynamics of SD, and hence being able to develop more effective measures to counter its spread, depends on the ability to characterise B. hyodysenteriae variants and trace relationships of epidemic strains. METHODOLOGY/PRINCIPAL FINDINGS: A collection of 51 Spanish and 1 Portuguese B. hyodysenteriae isolates was examined using a multilocus sequence typing (MLST) scheme based on the sequences of seven conserved genomic loci. The isolates were allocated to 10 sequence types (STs) in three major groups of descent. Isolates in four of the STs were widely distributed in farms around Spain. One farm was infected with isolates from more than one ST. Sequence data obtained from PubMLST for 111 other B. hyodysenteriae strains from other countries then were included in the analysis. Two of the predominant STs that were found in Spain also were present in other European countries. The 73 STs were arranged in eleven clonal complexes (Cc) containing between 2 and 26 isolates. A population snapshot based on amino acid types (AATs) placed 75% of the isolates from 32 of the 48 AATs into one major cluster. The founder type AAT9 included 22 isolates from 10 STs that were recovered in Spain, Australia, Sweden, Germany, Belgium, the UK, Canada, and the USA. CONCLUSIONS/SIGNIFICANCE: This MLST scheme provided sufficient resolution power to unambiguously characterise B. hyodysenteriae isolates, and can be recommended as a routine typing tool that rapidly enables comparisons of isolates. Using this method it was shown that some of the main genetic lineages of B. hyodysenteriae in Spain also occurred in other countries, providing further evidence for international transmission. Finally, analysis of AATs appeared useful for deducing putative ancestral relationships between strains.

A high dietary concentration of inulin is necessary to reduce the incidence of swine dysentery in pigs experimentally challenged with Brachyspira hyodysenteriae.

A total of sixty surgically castrated male pigs (Large White × Landrace) weighing 31·2 (sd 4·3) kg were used in a randomised block experiment to examine the effect of added dietary inulin (0, 20, 40 and 80 g/kg) on the occurrence of swine dysentery (SD) and on fermentation characteristics in the large intestine after experimental challenge with the causative spirochaete Brachyspira hyodysenteriae. The pigs were allowed to adapt to the diets for 2 weeks before each pig was challenged orally four times with a broth culture containing B. hyodysenteriae on consecutive days. Increasing dietary levels of inulin linearly (P = 0·001) reduced the risk of pigs developing SD; however, eight out of fifteen pigs fed the diet with 80 g/kg inulin still developed the disease. The pH values in the caecum (P = 0·072) tended to decrease, and in the upper colon, the pH values did decrease (P = 0·047) linearly with increasing inulin levels in the diets, most probably due to a linear increase in the concentration of total volatile fatty acids in the caecum (P = 0·018), upper colon (P = 0·001) and lower colon (P = 0·013). In addition, there was a linear reduction in the proportion of the branched-chain fatty acids isobutyric acid and isovaleric acid in the caecum (P = 0·015 and 0·026) and upper colon (P = 0·011 and 0·013) with increasing levels of dietary inulin. In conclusion, the present study showed that a diet supplemented with a high level of inulin (80 g/kg) but not lower levels reduced the risk of pigs developing SD, possibly acting through a modification of the microbial fermentation patterns in the large intestine.

Evidence that the 36 kb plasmid of Brachyspira hyodysenteriae contributes to virulence.

Swine dysentery (SD) results from infection of the porcine large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Recently the genome of virulent Australian B. hyodysenteriae strain WA1 was sequenced, and a 36 kilobase (kb) circular plasmid was identified. The plasmid contained 31 genes including six rfb genes that were predicted to be involved with rhamnose biosynthesis, and others associated with glycosylation. In the current study a set of PCRs was developed to amplify portions of nine of the plasmid genes. When used with DNA extracted from virulent strain B204, PCR products were generated, but no products were generated with DNA from avirulent strain A1. Analysis of the DNA using pulsed field gel electrophoresis (PFGE) identified a plasmid band in strains WA1 and B204, but not in strain A1. These results demonstrate that strain A1 does not contain the plasmid, and suggests that lack of the plasmid may explain why this strain is avirulent. To determine how commonly strains lacking plasmids occur, DNA was extracted from 264 Australian field isolates of B. hyodysenteriae and subjected to PCRs for three of the plasmid genes. Only one isolate (WA400) that lacked the plasmid was identified, and this absence was confirmed by PFGE analysis of DNA from the isolate and further PCR testing. To assess its virulence, 24 pigs were experimentally challenged with cultures of WA400, and 12 control pigs were challenged with virulent strain WA1 under the same conditions. Significantly fewer (P=0.03) of the pigs challenged with WA400 became colonised and developed SD (13/24; 54%) compared to the pigs infected with WA1 (11/12; 92%). Gross lesions in the pigs colonised with WA400 tended to be less extensive than those in pigs colonised with WA1, although there were no obvious differences at the microscopic level. The results support the likelihood that plasmid-encoded genes of B. hyodysenteriae are involved in colonisation and/or disease expression.

The complete genome sequence of the pathogenic intestinal spirochete Brachyspira pilosicoli and comparison with other Brachyspira genomes.

BACKGROUND: The anaerobic spirochete Brachyspira pilosicoli colonizes the large intestine of various species of birds and mammals, including humans. It causes "intestinal spirochetosis", a condition characterized by mild colitis, diarrhea and reduced growth. This study aimed to sequence and analyse the bacterial genome to investigate the genetic basis of its specialized ecology and virulence. METHODOLOGY/PRINCIPAL FINDINGS: The genome of B. pilosicoli 95/1000 was sequenced, assembled and compared with that of the pathogenic Brachyspira hyodysenteriae and a near-complete sequence of Brachyspira murdochii. The B. pilosicoli genome was circular, composed of 2,586,443 bp with a 27.9 mol% G+C content, and encoded 2,338 genes. The three Brachyspira species shared 1,087 genes and showed evidence of extensive genome rearrangements. Despite minor differences in predicted protein functional groups, the species had many similar features including core metabolic pathways. Genes distinguishing B. pilosicoli from B. hyodysenteriae included those for a previously undescribed bacteriophage that may be useful for genetic manipulation, for a glycine reductase complex allowing use of glycine whilst protecting from oxidative stress, and for aconitase and related enzymes in the incomplete TCA cycle, allowing glutamate synthesis and function of the cycle during oxidative stress. B. pilosicoli had substantially fewer methyl-accepting chemotaxis genes than B. hyodysenteriae and hence these species are likely to have different chemotactic responses that may help to explain their different host range and colonization sites. B. pilosicoli lacked the gene for a new putative hemolysin identified in B. hyodysenteriae WA1. Both B. pilosicoli and B. murdochii lacked the rfbBADC gene cluster found on the B. hyodysenteriae plasmid, and hence were predicted to have different lipooligosaccharide structures. Overall, B. pilosicoli 95/1000 had a variety of genes potentially contributing to virulence. CONCLUSIONS/SIGNIFICANCE: The availability of the complete genome sequence of B. pilosicoli 95/1000 will facilitate functional genomics studies aimed at elucidating host-pathogen interactions and virulence.

Multiple-locus variable-number tandem-repeat analysis of the swine dysentery pathogen, Brachyspira hyodysenteriae.

The spirochete Brachyspira hyodysenteriae is the causative agent of swine dysentery, a severe colonic infection of pigs that has a considerable economic impact in many swine-producing countries. In spite of its importance, knowledge about the global epidemiology and population structure of B. hyodysenteriae is limited. Progress in this area has been hampered by the lack of a low-cost, portable, and discriminatory method for strain typing. The aim of the current study was to develop and test a multiple-locus variable-number tandem-repeat analysis (MLVA) method that could be used in basic veterinary diagnostic microbiology laboratories equipped with PCR technology or in more advanced laboratories with access to capillary electrophoresis. Based on eight loci, and when performed on isolates from different farms in different countries, as well as type and reference strains, the MLVA technique developed was highly discriminatory (Hunter and Gaston discriminatory index, 0.938 [95% confidence interval, 0.9175 to 0.9584]) while retaining a high phylogenetic value. Using the technique, the species was shown to be diverse (44 MLVA types from 172 isolates and strains), although isolates were stable in herds over time. The population structure appeared to be clonal. The finding of B. hyodysenteriae MLVA type 3 in piggeries in three European countries, as well as other, related, strains in different countries, suggests that spreading of the pathogen via carrier pigs is likely. MLVA overcame drawbacks associated with previous typing techniques for B. hyodysenteriae and was a powerful method for epidemiologic and population structure studies on this important pathogenic spirochete.

Brachyspira intermedia strain diversity and relationships to the other indole-positive Brachyspira species.

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.

Multilocus sequence typing as a tool for studying the molecular epidemiology and population structure of Brachyspira hyodysenteriae.

The purpose of this study was to develop and apply a multilocus sequence typing (MLST) scheme to study the molecular epidemiology of Brachyspira hyodysenteriae, the aetiological agent of swine dysentery. Sequences of seven conserved genomic loci were examined in 111 B. hyodysenteriae strains. Fifty-eight of these previously had been analysed by multilocus enzyme electrophoresis (MLEE), and for some the results of pulsed field gel electrophoresis (PFGE), restriction endonuclease analysis (REA) and/or serotyping also were available. The discriminatory power of these methods was compared. The strains were divided into 67 sequence types (STs) and 46 amino acid types (AATs) by MLST. The Index of Association value was significantly different from zero, indication that the population was clonal. Eleven clonal complexes (Cc) comprising between 2 and 10 STs were recognised. A population snapshot based on AATs placed 77.5% of the isolates from 30 of the AATs into one major cluster. The founder type AAT9 included 13 strains from nine STs that were isolated in Australia, Sweden, Germany and Belgium, including one from a mallard. The MLST results were generally comparable to those produced by MLEE. The MLST system had a similar discriminatory power to PFGE, but was more discriminatory than REA, MLEE or serotyping. MLST data provided evidence for likely transmission of strains between farms, but also for the occurrence of temporal "micro-evolution" of strains on individual farms. Overall, the MLST system proved to be a useful new tool for investigating the molecular epidemiology and diversity of B. hyodysenteriae.

Genome sequence of the pathogenic intestinal spirochete brachyspira hyodysenteriae reveals adaptations to its lifestyle in the porcine large intestine.

Brachyspira hyodysenteriae is an anaerobic intestinal spirochete that colonizes the large intestine of pigs and causes swine dysentery, a disease of significant economic importance. The genome sequence of B. hyodysenteriae strain WA1 was determined, making it the first representative of the genus Brachyspira to be sequenced, and the seventeenth spirochete genome to be reported. The genome consisted of a circular 3,000,694 base pair (bp) chromosome, and a 35,940 bp circular plasmid that has not previously been described. The spirochete had 2,122 protein-coding sequences. Of the predicted proteins, more had similarities to proteins of the enteric Escherichia coli and Clostridium species than they did to proteins of other spirochetes. Many of these genes were associated with transport and metabolism, and they may have been gradually acquired through horizontal gene transfer in the environment of the large intestine. A reconstruction of central metabolic pathways identified a complete set of coding sequences for glycolysis, gluconeogenesis, a non-oxidative pentose phosphate pathway, nucleotide metabolism, lipooligosaccharide biosynthesis, and a respiratory electron transport chain. A notable finding was the presence on the plasmid of the genes involved in rhamnose biosynthesis. Potential virulence genes included those for 15 proteases and six hemolysins. Other adaptations to an enteric lifestyle included the presence of large numbers of genes associated with chemotaxis and motility. B. hyodysenteriae has diverged from other spirochetes in the process of accommodating to its habitat in the porcine large intestine.

A reverse vaccinology approach to swine dysentery vaccine development.

Swine dysentery (SD) is a mucohaemorrhagic colitis of pigs resulting from infection of the large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Whole-cell bacterin vaccines are available to help control SD, but their performance has been inconsistent. This study aimed to use a reverse vaccinology approach to identify B. hyodysenteriae proteins for use as recombinant vaccine components. Nineteen open reading frames (ORFs) predicted to encode potential vaccine candidate molecules were identified from in silico analysis of partial genomic sequence data. The distribution of these ORFs among strains of B. hyodysenteriae was investigated by PCR, and widely distributed ORFs were cloned. The products were screened with a panel of immune pig sera, and from these a subset of conserved, immunogenic proteins was selected. Mice immunized intramuscularly with these recombinant proteins developed specific systemic antibody responses to them, and their sera agglutinated B. hyodysenteriae cells in vitro. In a pilot experiment, eight pigs were vaccinated twice intramuscularly with a combination of four of the proteins. The pigs developed antibodies to the proteins, and following experimental challenge only one developed SD compared to five of nine non-vaccinated control pigs. Although these differences in incidence were not significant, they indicated a trend towards protection using the recombinant proteins as immunogens. This study demonstrates that the reverse vaccinology approach has considerable potential for use in developing novel recombinant vaccines for SD.