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 duplex PCR (D-PCR) amplifying portions of the Brachyspira hyodysenteriae NADH oxidase gene and the B. pilosicoli 16S rRNA gene was developed and then tested on DNA extracted from 178 porcine fecal samples. The feces also underwent anaerobic culture and species-specific PCRs. Fecal extraction-D-PCR detected seven additional samples containing B. hyodysenteriae and five more containing B. pilosicoli.Pigs are colonized by two pathogenic species of anaerobic intestinal spirochetes, Brachyspira hyodysenteriae and B. pilosicoli. B. hyodysenteriae causes swine dysentery, a severe mucohemorrhagic diarrheal disease (10), while B. pilosicoli causes porcine intestinal spirochetosis, a milder colitis that also occurs in other host species (9). Because of the economic impact of these diseases on pig production, rapid diagnostic methods are needed to detect and distinguish between them. Historically, a positive diagnosis of swine dysentery or porcine intestinal spirochetosis has required isolation of the associated spirochete and confirmation of its identity by performance of phenotypic tests (11). However, the fastidious and slow-growing nature of intestinal spirochetes, together with the limited range of phenotypic differences between them, has hampered this approach. To overcome such problems, a variety of PCR assays for B. hyodysenteriae and B. pilosicoli have been developed (1-3, 7, 8, 14, 15). These PCRs target conserved genes such as the 16S rRNA gene (rDNA), the 23S rDNA, the NADH oxidase (nox) gene, or the hemolysin (tly) gene. The assays typically are conducted on intestinal spirochete isolates or on spirochetal growth harvested from primary isolation plates. Difficulties have been encountered when trying to extract spirochetal DNA directly from feces for use as a PCR template, and there appears to be only one report of the use of PCR (plus hybridization) for direct detection of B. hyodysenteriae from feces (5) and one recent report of its use for B. pilosicoli (4).A multiplex PCR for the simultaneous detection of B. hyodysenteriae, Lawsonia intracellularis (the agent of porcine proliferative enteropathy), and Salmonella spp. has been described previously (6), but to date, no PCRs amplifying and distinguishing between B. hyodysenteriae and B. pilosicoli in one reaction mixture have been reported. The purpose of the present study was to develop a reliable and robust duplex PCR (D-PCR) system that can be used to detect these two species by using DNA extracted directly from pig feces. To evaluate the test as a diagnostic tool, it was compared with selective anaerobic culture, followed by individual species-specific PCRs conducted on growth harvested from the primary isolation plates.Control spirochete strains and culture conditions. Control spirochete strains were obtained from the culture collection held at the Reference Centre for Intestinal Spirochetes, Murdoch University, Western Australia, Australia. These included the type strains of B. hyodysenteriae (B78 T ) and B. pilosicoli (P43/6/78 T ), which were used to...

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The purpose of this study was to develop and apply a multilocus sequence typing 27 (MLST) scheme to study the molecular epidemiology of Brachyspira hyodysenteriae, 28 the aetiological agent of swine dysentery. Sequences of seven conserved genomic loci 29 were examined in 111 B. hyodysenteriae strains. Fifty-eight of these previously had 30 been analysed by multilocus enzyme electrophoresis (MLEE), and for some the 31 results of pulsed field gel electrophoresis (PFGE), restriction endonuclease analysis 32 (REA) and/or serotyping also were available. The discriminatory power of these 33 methods was compared. The strains were divided into 67 sequence types (STs) and 46 34 amino acid types (AATs) by MLST. The Index of Association value was significantly 35 different from zero, indication that the population was clonal. Eleven clonal 36 complexes (Cc) comprising between 2 and 10 STs were recognised. A population 37 snapshot based on AATs placed 77.5% of the isolates from 30 of the AATs into one 38 major cluster. The founder type AAT9 included 13 strains from nine STs that were 39 isolated in Australia, Sweden, Germany and Belgium, including one from a mallard. 40The MLST results were generally comparable to those produced by MLEE. The 41 MLST system had a similar discriminatory power to PFGE, but was more 42 discriminatory than REA, MLEE or serotyping. MLST data provided evidence for 43 likely transmission of strains between farms, but also for the occurrence of temporal 44 "micro-evolution" of strains on individual farms. Overall, the MLST system proved to 45 be a useful new tool for investigating the molecular epidemiology and diversity of B. 46 hyodysenteriae. 47 48

BackgroundThe 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 FindingsThe 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/SignificanceThe availability of the complete genome sequence of B. pilosicoli 95/1000 will facilitate functional genomics studies aimed at elucidating host-pathogen interactions and virulence.

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.Avian intestinal spirochetosis (AIS) is a disease complex that affects commercial laying and meat breeder chickens, characterized by the colonization of the ceca by members of one or more species of anaerobic intestinal spirochetes of the genus Brachyspira (formerly Serpulina) (11,14,36). The condition is associated with delayed or reduced egg production and a chronic diarrhea in adult birds that can result in wet litter and fecal staining of eggshells. Moreover, reduced performance has been reported for broiler chicks hatched from eggs of breeder chickens infected with Brachyspira spp. (7,33). AIS was first clearly described in the 1980s, in both The Netherlands (5, 7, 8) and the United Kingdom (10), and subsequently has been reported in the United States (39, 43), Australia (24,35), and a number of other European countries (2, 3). The nonspecific nature of the disease and the lack of availability of simple and specific diagnostic tests for these anaerobic spirochetes from chickens may have resulted in the condition being greatly underreported (14).Studies using multilocus enzyme electrophoresis showed that intestinal spirochetes from chickens could be divided into at least six species groupings (25). These included the three species currently considered to be pathogenic to chickens, Brachys...