First identification and characterisation of Brachyspira hyodysenteriae in pigs in Hong Kong.

Swine dysentery (SD) is an important endemic disease of pigs throughout the world. The most common aetiological agent is the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. The related spirochaete Brachyspira pilosicoli causes a milder form of colitis. We report the first isolation of B. hyodysenteriae and B. pilosicoli from a pig farm in Hong Kong. Faecal samples containing mucus or fresh blood were collected from the ground where finisher pigs had just been loaded into a truck for transport to the abattoir. The samples were subjected to selective anaerobic culture and PCR for B. hyodysenteriae and B. pilosicoli, and two isolates of both species were obtained. The B. hyodysenteriae isolates showed clinical resistance to tylosin and lincomycin, whilst the B. pilosicoli isolates were resistant to tylosin and showed intermediate susceptibility to lincomycin. The B. hyodysenteriae isolates were subjected to multilocus sequence typing and a single previously undescribed sequence type (ST250) was identified. Disease was not recorded in other pigs on the farm, but it may have been masked by the use of antimicrobials. Further work is required to examine the distribution of these two pathogens in this and other farms in Hong Kong and in adjoining mainland China.

Identification of Brachyspira species by cpn60 universal target sequencing is superior to NADH oxidase gene sequencing.

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.

Correction to: The pathogenic intestinal spirochaete Brachyspira pilosicoli forms a diverse recombinant species demonstrating some local clustering of related strains and potential for zoonotic spread.

[This corrects the article DOI: 10.1186/1757-4749-5-24.].

An atypical weakly haemolytic strain of Brachyspira hyodysenteriae is avirulent and can be used to protect pigs from developing swine dysentery.

The anaerobic intestinal spirochaete Brachyspira hyodysenteriae colonises the large intestine of pigs and causes swine dysentery (SD), a severe mucohaemorrhagic colitis. SD occurs worldwide, and control is hampered by a lack of vaccines and increasing antimicrobial resistance. B. hyodysenteriae strains typically produce strong beta-haemolysis on blood agar, and the haemolytic activity is thought to contribute to the pathogenesis of SD. Recently, weakly haemolytic variants of B. hyodysenteriae have been identified in Europe and Australia, and weakly haemolytic strain D28 from Belgium failed to cause disease when used experimentally to infect pigs. Moreover, pigs colonised with D28 and then challenged with virulent strongly haemolytic strain B204 showed a delay of 2-4 days in developing SD compared to pigs not exposed to D28. The current study aimed to determine whether Australian weakly haemolytic B. hyodysenteriae strain MU1, which is genetically distinct from D28, could cause disease and whether exposure to it protected pigs from subsequent challenge with strongly haemolytic virulent strains. Three experimental infection studies were undertaken in which no diseases occurred in 34 pigs inoculated with MU1, although mild superficial lesions were found in the colon in 2 pigs in one experiment. In two experiments, significantly fewer pigs exposed to MU1 and then challenged with strongly haemolytic virulent strains of B. hyodysenteriae developed SD compared to control pigs not previously exposed to MU1 (p = 0.009 and p = 0.0006). These data indicate that MU1 lacks virulence and has potential to be used to help protect pigs from SD.

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.

Antimicrobial resistance in Brachyspira - An increasing problem for disease control.

Across all bacterial species the continuing reduction in susceptibility to antimicrobial agents is a critical and increasing threat for disease control. This mini-review outlines the extent of this problem amongst anaerobic intestinal spirochaetes of the genus Brachyspira, of which there are currently nine officially recognised species. These include some important pathogens that may cause colitis with diarrhoea and/or dysentery in various mammalian and avian species, but most notably in pigs and in adult chickens. The most economically significant pathogen is Brachyspira hyodysenteriae, the spirochaete which causes swine dysentery in countries throughout the world. Control of infections with Brachyspira species has long relied on the prophylactic or therapeutic use of antimicrobials, but increasingly strains with reduced susceptibility and sometimes multidrug resistance to previously effective antimicrobial agents are being encountered. In this mini-review we outline these problems and explain the extent and molecular basis of the emerging resistance. Future control will rely on developing and applying standardised methods for measuring antimicrobial susceptibility; improving surveillance of resistance using traditional phenotypic as well as genomic analysis of known resistance determinants; improving understanding of the molecular basis of resistance to different drug classes; improving farmer and veterinarian education about prudent antimicrobial use so as to reduce selective pressure on the emergence of resistance; and developing alternatives to antimicrobials as a means to control these infections.

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

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

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).

Comparison of Brachyspira hyodysenteriae Isolates Recovered from Pigs in Apparently Healthy Multiplier Herds with Isolates from Herds with Swine Dysentery.

Swine dysentery (SD) is a mucohaemorrhagic colitis of grower/finisher pigs classically resulting from infection by the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. This study aimed to determine whether B. hyodysenteriae isolates from pigs in three healthy German multiplier herds supplying gilts to other farms differed from isolates from nine German production herds with SD. Isolates were subjected to whole genomic sequencing, and in silico multilocus sequence typing showed that those from the three multiplier herds were of previously undescribed sequence types (ST132, ST133 and ST134), with all isolates from the same herd having the same ST. All isolates were examined for the presence of 332 genes encoding predicted virulence or virulence lifestyle associated factors, and these were well conserved. Isolates from one multiplier herd were atypical in being weakly haemolytic: they had 10 amino acid substitutions in the haemolysin III protein and five in the haemolysin activation protein compared to reference strain WA1, and had a disruption in the promoter site of the hlyA gene. These changes likely contribute to the weakly haemolytic phenotype and putative lack of virulence. These same isolates also had nine base pair insertions in the iron metabolism genes bitB and bitC and lacked five of six plasmid genes that previously have been associated with colonisation. Other overall differences between isolates from the different herds were in genes from three of five outer membrane proteins, which were not found in all the isolates, and in members of a block of six plasmid genes. Isolates from three herds with SD had all six plasmid genes, while isolates lacking some of these genes were found in the three healthy herds-but also in isolates from six herds with SD. Other differences in genes of unknown function or in gene expression may contribute to variation in virulence; alternatively, superior husbandry and better general health may have made pigs in the two multiplier herds colonised by "typical" strongly haemolytic isolates less susceptible to disease expression.

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.

Analysis of Multiple Brachyspira hyodysenteriae Genomes Confirms That the Species Is Relatively Conserved but Has Potentially Important Strain Variation.

The intestinal spirochete Brachyspira hyodysenteriae is an important pathogen in swine, causing mucohemorrhagic colitis in a disease known as swine dysentery. Based on the detection of significant linkage disequilibrium in multilocus sequence data, the species is considered to be clonal. An analysis of the genome sequence of Western Australian B. hyodysenteriae strain WA1 has been published, and in the current study 19 further strains from countries around the world were sequenced with Illumina technology. The genomes were assembled and aligned to over 97.5% of the reference WA1 genome at a percentage sequence identity better than 80%. Strain regions not aligned to the reference ranged between 0.2 and 2.5%. Clustering of the strain genes found on average 2,354 (88%) core genes, 255 (8.6%) ancillary genes and 77 (2.9%) unique genes per strain. Depending on the strain the proportion of genes with 100% sequence identity to WA1 ranged from 85% to 20%. The result is a global comparative genomic analysis of B. hyodysenteriae genomes revealing potential differential phenotypic markers for numerous strains. Despite the differences found, the genomes were less varied than those of the related pathogenic species Brachyspira pilosicoli, and the analysis supports the clonal nature of the species. From this study, a public genome resource has been created that will serve as a repository for further genetic and phenotypic studies of these important porcine bacteria. This is the first intra-species B. hyodysenteriae comparative genomic analysis.

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.

The pathogenic intestinal spirochaete Brachyspira pilosicoli forms a diverse recombinant species demonstrating some local clustering of related strains and potential for zoonotic spread.

BACKGROUND: Brachyspira pilosicoli is an anaerobic spirochaete that can colonizes the large intestine of many host species. Infection is particularly problematic in pigs and adult poultry, causing colitis and diarrhea, but it is also known to result in clinical problems in human beings. Despite the economic importance of the spirochaete as an animal pathogen, and its potential as a zoonotic agent, it has not received extensive study. METHODS: A multilocus sequence typing (MLST) method based on the scheme used for other Brachyspira species was applied to 131 B. pilosicoli isolates originating from different host species and geographical areas. A variety of phylogenetic trees were constructed and analyzed to help understand the data. RESULTS: The isolates were highly diverse, with 127 sequence types and 123 amino acid types being identified. Large numbers (50-112) of alleles were present at each locus, with all loci being highly polymorphic. The results of Shimodaira-Hasegawa tests identified extensive genetic recombination, although the calculated standardized index of association value (0.1568; P <0.0005) suggested the existence of some clonality. Strains from different host species and geographical origins generally were widely distributed throughout the population, although in nine of the ten cases where small clusters of related isolates occurred these were from the same geographical areas or farms/communities, and from the same species of origin. An exception to the latter was a cluster of Australian isolates originating from pigs, chickens and a human being, suggesting the likelihood of relatively recent transmission of members of this clonal group between species. CONCLUSIONS: The strongly recombinant population structure of B. pilosicoli contrasts to the more highly clonal population structures of the related species Brachyspira hyodysenteriae and Brachyspira intermedia, both of which are specialized enteric pathogens of pigs and poultry. The genomic plasticity of B. pilosicoli may help to explain why it has been able to adapt to colonize the large intestines of a wider range of hosts compared to other Brachyspira species. The identification of a clonal group of isolates that had been recovered from different host species, including a human being, suggests that zoonotic transmission by B. pilosicoli may occur in nature. Evidence for local transmission between the same host species also was obtained.

Multiple locus variable number tandem repeat analysis (MLVA) of the pathogenic intestinal spirochaete Brachyspira pilosicoli.

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.