The frequency of tail damage amongst cows from a sample of New Zealand dairy farms participating in an animal welfare programme.

AIM: To explore factors associated with the frequency of tail damage in dairy cows on 29 New Zealand farms participating in an animal welfare monitoring programme. MATERIALS AND METHODS: Herd-level tail score data were collected at the cow level and then summarised at the herd level as counts for each lactation over the period 1 June 2014 to 31 May 2018. A cow's tail was considered damaged if there was evidence of any injury that deformed the anatomical structure involving either bone or soft tissue and could include loss of use. There were four categories for tail scoring. Fracture or dislocation of tail bones was considered as a deviation (score 1). When the tail had been docked above the top of the cow's udder, this was considered as docked short tail (score 2). When there was evidence of soft tissue trauma (score 3) or bone damage but no fracture (score 4), this was recorded as damaged (other). Tails were scored for each whole dairy herd. Tail scoring was performed by trained veterinarians or veterinary technicians. The primary outcome variable was counts of deviated tails (DT). Other outcome variables were docked short, damaged (other), and total tail injuries (TTI) which was a summation of all tail injuries. The potential predictor variables were area, season, farm, region, replacement rate, and herd size. A mixed-effects negative binomial or Poisson regression was fitted to the count data. RESULTS: A total of 29 farms contributed data for tail scoring, with 54,831 cows individually scored. The unadjusted regional prevalence of TTI ranged from 3.5% (64/1,835) in Taranaki in 2014-2015 to 28.7% (1,434/4,988) in Southland/Otago in 2017-2018. The unadjusted regional herd prevalence of DT ranged from 2.1% (280/6,862) in Taranaki (2014-2015) to 13.2% (4,627/30,165) in Southland/South Otago (2017-2018). The incident rate ratio (IRR) of DT in 2015-2016 was 1.74 (95% CI = 1.20-2.53; p = 0.003) times the incident rate for the reference group (2014-2015). The IRR for TTI in 2015-2016 was 1.70 (95% CI = 1.60-1.81; p = 0.001) times the incident rate for the reference group (2014-2015). CONCLUSIONS AND CLINICAL RELEVANCE: This is the first quantitative study of the frequency of tail damage within New Zealand dairy farms and whilst variable between regions, it indicates that the frequency is increasing. Opportunities exist to better understand the causes of tail injuries and to improve animal welfare.

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

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

Gastrointestinal health and function in weaned pigs: a review of feeding strategies to control post-weaning diarrhoea without using in-feed antimicrobial compounds.

For the last several decades, antimicrobial compounds have been used to promote piglet growth at weaning through the prevention of subclinical and clinical disease. There are, however, increasing concerns in relation to the development of antibiotic-resistant bacterial strains and the potential of these and associated resistance genes to impact on human health. As a consequence, European Union (EU) banned the use of antibiotics as growth promoters in swine and livestock production on 1 January 2006. Furthermore, minerals such as zinc (Zn) and copper (Cu) are not feasible alternatives/replacements to antibiotics because their excretion is a possible threat to the environment. Consequently, there is a need to develop feeding programs to serve as a means for controlling problems associated with the weaning transition without using antimicrobial compounds. This review, therefore, is focused on some of nutritional strategies that are known to improve structure and function of gastrointestinal tract and (or) promote post-weaning growth with special emphasis on probiotics, prebiotics, organic acids, trace minerals and dietary protein source and level.

Development of a modified selective medium to enhance the recovery rate of Brachyspira hyodysenteriae and other porcine intestinal spirochaetes from faeces.

AIMS: The aim of this study was to develop a modified selective medium to improve the recovery rate of Brachyspira hyodysenteriae and other clinically significant intestinal spirochaetes from porcine faeces. METHODS AND RESULTS: The susceptibility of five Brachyspira spp. type strains and five Thai field isolates of B. hyodysenteriae to the antimicrobials halquinol and flavomycin was determined by in vitro susceptibility tests in the agar dilution method, and optimal incorporation rates were confirmed by broth dilution. All the spirochaetes were susceptible to halquinol at ≤ 1 µg ml(-1), while 16 µg ml(-1) of flavomycin (F) allowed their growth, and therefore, only the latter was selected for further use. F and different combinations of colistin (C), spectinomycin (S) and rifampacin (R) were incorporated into pre-enrichment broths and/or agar plates, and growth of the spirochaetes from seeded faeces was determined. Two solid media were selected for further testing using faeces from 90 finishing pigs on 10 farms. A previously recommended method of pre-enrichment did not increase the recovery rate. The use of blood agar modified medium (BAM) containing F (16 µg ml(-1)), S (400 µg ml(-1)), R (30 µg ml(-1)) and colistin (C, 100 U ml(-1)) (assigning as BAM-CSRF) reduced the growth of contaminating intestinal microbiota and resulted in a significantly higher rate of spirochaete recovery than the previous recommended medium. CONCLUSION: BAM-CSRF is a useful new selective medium for the isolation of B. hyodysenteriae and other intestinal spirochaetes from pig faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The new selective medium for isolating B. hyodysenteriae and other Brachyspira spp. from pig faeces will improve their recovery and subsequent disease diagnosis.

Understanding the molecular epidemiology of the footrot pathogen Dichelobacter nodosus to support control and eradication programs.

The Gram-negative anaerobe Dichelobacter nodosus is the primary etiologic agent of ovine footrot. Few studies of the genetic diversity and epidemiology of D. nodosus have been done, despite the economic cost and welfare implications of the disease. This study examined a large collection of Australian isolates; 735 isolates from footrot-infected sheep from 247 farms in Western Australia (WA) were tested by pulsed-field gel electrophoresis (PFGE), and a subset of 616 isolates was tested by infrequent restriction site PCR (IRS-PCR). The genetic diversity of WA isolates was compared to that of 61 isolates from three other Australian states. WA isolates were genetically diverse, with 181 molecular types resolved by PFGE, resulting in a simple diversity ratio (SDR) of 1:4 and a Simpson's index of discrimination value (D) of 0.98. IRS-PCR resolved 77 molecular types (SDR = 1:8 and D = 0.95). The isolates were grouped into 67 clonal groups by PFGE (SDR = 1:11, D = 0.90) and 36 clonal groups by IRS-PCR (SDR = 1:17, D = 0.87). Despite the high genetic diversity, three common clonal groups predominated in WA and were found in other Australian states. On some farms, molecular type was stable over a number of years, whereas on other farms genetically diverse isolates occurred within a flock of sheep or within a hoof. This study provides a large database from which to appropriately interpret molecular types found in epidemiological investigations and to identify common and unknown types that may compromise footrot eradication or control programs.

Testing the efficacy of kitasamycin for use in the control and treatment of swine dysentery in experimentally infected pigs.

BACKGROUND: Swine dysentery (SD) caused by Brachyspira hyodysenteriae is an important disease in Australia. AIM: The aim of this study is to evaluate the macrolide antibiotic kitasamycin for use in SD control. METHODS: The minimum inhibitory concentrations (MICs) of kitasamycin, tylosin and lincomycin for 32 Australian isolates of B. hyodysenteriae were evaluated. Mutations in the 23S rRNA gene were examined. Isolate '13' with a low kitasamycin MIC was used to challenge weaner pigs. Sixty pigs were housed in 20 pens each containing three pigs: pigs in four pens received 2 kg/tonne of a product containing kitasamycin (3.1% active) prophylactically in their food starting 4 days before B. hyodysenteriae challenge (group 1); pigs in four pens were challenged and received the same dose therapeutically once one pig in a pen showed diarrhoea (group 2); four pens were challenged and received 4 kg/tonne of the product therapeutically (group 3); four pens were challenged but not medicated (group 4); two pens were unmedicated and unchallenged (group 5) and two pens received 2 kg/tonne and were unchallenged (group 6). Pigs were monitored for B. hyodysenteriae excretion and disease. RESULTS: Macrolide resistance was widespread, and mutations in the 23S rRNA gene were identified in 23 isolates. Four isolates with kitasamycin MICs < 5 µg/mL were considered susceptible. Following experimental challenge, 10 of 12 unmedicated pigs developed SD. No pigs receiving kitasamycin prophylactical or therapeutically developed SD. Medicated pigs shed low numbers of B. hyodysenteriae in their faeces. CONCLUSIONS: Kitasamycin can help control SD in pigs infected with susceptible isolates of B. hyodysenteriae.

Prevalence, disease associations and risk factors for colonization with intestinal spirochaetes (Brachyspira spp.) in flocks of laying hens in north-eastern Italy.

The present study investigated the occurrence of anaerobic intestinal spirochaetes of the genus Brachyspira in laying hen flocks in Treviso province, north-eastern Italy, with respect to prevalence, spirochaete species present, disease associations and risk factors for colonization. A total of 450 faecal samples from 45 sheds on 29 laying hen farms were cultured for intestinal spirochaetes. Nineteen sheds on 12 farms contained chickens with symptoms consistent with avian intestinal spirochaetosis, including reduced egg production, wet litter and/or pasty vents. Spirochaetes were isolated from 157 (34.8%) samples from 21 (72.4%) farms, and from 32 (71.1%) sheds. From these positive samples, 189 spirochaetal isolates were speciated using three polymerase chain reaction assays and a restriction fragment polymorphism analysis of 16S rDNA polymerase chain reaction products. Overall, 52 (27.5%) isolates were identified as pathogenic Brachyspira intermedia, 26 (13.8%) as pathogenic Brachyspira pilosicoli, 93 (49.7%) as non-pathogenic (Brachyspira innocens/Brachyspira murdochii), and 18 (9.6%) were unidentified. Faeces from 14 sheds (31%) on 10 farms (34.5%) contained B. intermedia and/or B. pilosicoli, and disease consistent with avian intestinal spirochaetosis was observed in nine of these sheds on seven farms. There was a significant association (P=0.042) between the presence of spirochaetes and using deep pits rather than conveyor belts for manure disposal. Sheds housing chickens >40 weeks of age were significantly more likely to contain spirochaetes (P=0.048) and pathogenic species (P=007) than sheds housing younger chickens. A significant association (P=0.02) was found between infection with pathogenic spirochaetes and reduced egg production.

A cross-sectional study to investigate the occurrence and distribution of intestinal spirochaetes (Brachyspira spp.) in three flocks of laying hens.

A cross-sectional study was conducted on a commercial egg-producing farm with a history of wet litter. A total of 600 fresh caecal faecal samples were obtained from under cages of laying hens in three sheds each containing flocks of approximately 5400 hens. Samples were cultured for intestinal spirochaetes, and growth on the primary isolation plate was observed under a phase contrast microscope and subjected to PCRs specific for the intestinal spirochaetes Brachyspira intermedia and Brachyspira pilosicoli. Spirochaete isolates obtained in pure culture were assessed for their ability to cause haemolysis on blood agar and to produce indole, and were typed using pulsed field gel electrophoresis (PFGE). A 1250 base pair portion of the 16S rRNA gene of three B. intermedia and five unidentified isolates was sequenced, and the sequences compared with those of other Brachyspira species. Overall, 121 (20.2%) of the faecal samples contained spirochaetes as determined by growth on the plate and microscopy. Using PCR on the primary growth from these positive samples, 43 (7.2% overall) were shown to contain B. intermedia, 8 (1.3%) to contain B. pilosicoli, and 70 (11.7%) were PCR negative. Only 24 isolates of B. intermedia and five isolates of unknown species were obtained in pure culture. Comparative analysis of the 16S rRNA gene sequence identified the non-B. intermedia isolates as belonging to the proposed species "Brachyspira pulli". PFGE analysis of the B. intermedia strains identified them as having four major banding patterns. Individual patterns were found in hens from different flocks, suggesting cross-transmission of strains between flocks. No environmental sources of infection were identified. The youngest flock had a significantly lower level of colonisation with B. intermedia than the flock of intermediate age (P = 0.004), suggesting that following initial infection of individual young hens on this farm there was amplification and transmission of infection amongst members of the flock.

The distribution of bmpB, a gene encoding a 29.7 kDa lipoprotein with homology to MetQ, in Brachyspira hyodysenteriae and related species.

The distribution of the bmpB gene encoding BmpB, a 29.7 kDa outer membrane lipoprotein of the intestinal spirochaete Brachyspira hyodysenteriae, was investigated. Using PCR, the gene was detected in all the 48 strains of B. hyodysenteriae examined and in Brachyspira innocens strain B256T, but not in 11 other strains of B. innocens nor in 42 strains of other Brachyspira spp. The gene was sequenced from B. innocens strain B256T and from 11 strains of B. hyodysenteriae. The B. hyodysenteriae genes shared 97.9-100% nucleotide sequence similarity and had 97.5-99.5% similarity with the gene of B. innocens strain B256T. Southern hybridisation indicated that bmpB was present on a 1.9 kb HindIII fragment of the B. hyodysenteriae genome and on a 3.1 kb fragment of the B. innocens B256T genome. The B. innocens lipoprotein did not react in Western blots with monoclonal antibody BJL/SH1 that reacts with the B. hyodysenteriae lipoprotein. The difference in binding with the monoclonal antibody may reside in the replacement of a serine residue with a tyrosine residue at base position 210 in the lipoprotein from B. innocens B256T. Comparison of the BmpB amino acid sequence with sequences in the SWISS-PROT protein database indicated that it has 33.9-39.9% similarity with the d-methionine binding proteins (MetQ) of a number of pathogenic bacterial species. The bmpB gene was confirmed to be the same as a gene of B. hyodysenteriae that was recently designated "blpA".

Sequence types and pleuromutilin susceptibility of Brachyspira hyodysenteriae isolates from Italian pigs with swine dysentery: 2003-2012.

Swine dysentery is a mucohaemorrhagic colitis of pigs caused by infection with Brachyspira hyodysenteriae. The disease can be controlled by treatment with antimicrobial agents, with the pleuromutilins tiamulin and valnemulin being widely used. In recent years, the occurrence of B. hyodysenteriae with reduced susceptibility to these drugs has been increasing. The aim of this study was to determine temporal changes in genetic groups and pleuromutilin susceptibility amongst B. hyodysenteriae isolates from Italy. Multilocus sequence typing (MLST) was performed on 108 isolates recovered from 87 farms in different regions of Italy from 2003 to 2012, and their minimum inhibitory concentrations (MICs) for tiamulin and valnemulin were determined. Logistic regression was performed to assess associations between susceptibility to the two antimicrobial agents and genetic group, year and region of isolation. The isolates were allocated to 23 sequence types (STs), with five clonal clusters (Ccs) and seven singletons. More than 50% of isolates were resistant to both pleuromutilins (MIC >2.0 µg/mL for tiamulin and >1.0 µg/mL for valnemulin). All 10 isolates in ST 83 were resistant; these were first isolated in 2011 and came from nine farms, suggesting recent widespread dissemination of a resistant strain. Significant associations were found between the proportion of pleuromutilin susceptible isolates and the genetic group and year of isolation. Although resistant isolates were found in all Ccs, isolates in Ccs 2 and 7 were over five times more likely to be susceptible than those in the other Ccs. A significant trend in the reduction of susceptibility over time also was observed.

A monoclonal antibody reacting with the cell envelope of spirochaetes isolated from cases of intestinal spirochaetosis in pigs and humans.

A monoclonal antibody (mAb) designed BJL/AC1 was prepared against the cell envelope of an intestinal spirochaete (strain 3295) that was isolated from a pig with intestinal spirochaetosis. The mAb reacted with a band of approximately 29 kDa in cell envelope preparations from 13 porcine and 11 human spirochaetes isolated from cases of intestinal spirochaetosis, but did not react with preparations made from a range of other intestinal spirochaetes. Immunogold labelling demonstrated that the reactive epitope was located on the cell envelope of the strains causing intestinal spirochaetosis. The mAb was used in an indirect immunofluorescence test to detect spirochaetes in the faeces of pigs with experimentally induced intestinal spirochaetosis. The mAb should prove to be a useful reagent for detection and identification of spirochaetes that are specifically associated with intestinal spirochaetosis.

Production and characterisation of a monoclonal antibody to Serpulina hyodysenteriae.

A monoclonal antibody (mAb) directed against Serpulina hyodysenteriae, the causative agent of swine dysentery, was produced and characterised. The mAb (BJL/SH1) reacted in Western blots with a protein with a molecular mass of about 30 kDa in outer membrane preparations from a range of S. hyodysenteriae isolates of different serotypes. It did not react with preparations made from a variety of non-S. hyodysenteriae intestinal spirochaetes. Immunogold labelling was used to confirm the location of the reactive epitope on the cell outer membrane. The mAb agglutinated and produced fluorescence only with strains of S. hyodysenteriae, and should prove to be a useful reagent for identification of S. hyodysenteriae.

Pulsed-field gel electrophoresis for sub-specific differentiation of Serpulina pilosicoli (formerly 'Anguillina coli').

Pulsed-field gel electrophoresis (PFGE) was developed for subspecific differentiation of Serpulina pilosicoli, and was applied to 52 isolates recovered from cases of intestinal spirochaetosis (IS) in pigs, dogs, human beings and various avian species. The technique was highly sensitive, differentiating the isolates into 40 groupings. Only six groups contained more than one isolate; in five of these groups isolates with the same banding pattern were either from pigs in the same herds (four groups), or from humans in the same community: the sixth group contained two identical Australian porcine isolates from unrelated herds in different states. Overall S. pilosicoli isolates were genetically diverse, but in some cases isolates cultured from the same or different animal species were closely related. This suggested the likelihood of cross-species transmission, including zoonotic spread. PFGE was a powerful tool for epidemiological studies of S. pilosicoli and also allowed examination of genetic relationships between isolates.

Polymerase chain reaction for identification of human and porcine spirochaetes recovered from cases of intestinal spirochaetosis.

A polymerase chain reaction (PCR) amplification of 16S rDNA was developed to identify spirochaetes recovered from cases of intestinal spirochaetosis in humans and pigs; these bacteria belong to a distinct genetic group of spirochaetes, with the proposed name 'Anguillina coli'. The PCR incorporated a universal eubacterial 16S rDNA sequencing primer (1492r), and a 21-base forward primer designed to include a nucleotide sequence specific for 'A. coli'. The PCR was used to correctly identify DNA extracted from 43 isolates of 'A. coli' from humans and pigs, whilst no product was produced from Escherichia coli, or from other intestinal spirochaetes, including 38 isolates of Serpulina spp., and one each of Treponema succinifaciens and Brachyspira aalborgi. The amplification provided a rapid and simple means of identifying DNA from isolates of 'A. coli', and could be used on boiled whole 'A. coli' cells, with a detection limit equivalent to 2.5 x 10(2) cells. The reaction was used to detect and identify these spirochaetes from selective agar plates inoculated with stool specimens from infected pigs.

Phenotypic characteristics of Serpulina pilosicoli the agent of intestinal spirochaetosis.

The phenotypic characteristics of three Serpulina pilosicoli strains isolated from humans with diarrhoea (WesB, Kar, Hrm7) and two porcine S. pilosicoli strains isolated from pigs with intestinal spirochaetosis (1648, 3295), were compared with the type strain of the species P43/6/78T (T = type strain) and other intestinal spirochaetes within the genus Serpulina. All S. pilosicoli strains had a characteristic ultrastructural appearance, displayed similar growth rates, hydrolysed hippurate, lacked beta-glucosidase activity, utilised D-ribose as a growth substrate, and had similar sensitivities to rifampicin and spiramycin. The only consistent phenotypic characteristic that differentiated human strains from porcine strains of S. pilosicoli was that the human strains all utilised the pentose sugar D-xylose. These distinguishing phenotypic traits appear useful for identifying S. pilosicoli.

Identification of a gene sequence encoding a putative pyruvate oxidoreductase in Serpulina pilosicoli.

Serpulina pilosicoli is a recently described species of intestinal spirochaete which can be identified using a species-specific monoclonal antibody BJL/AC1 reactive with a 29-kDa protein located in the cell envelope. A genomic library of the type strain of S. pilosicoli P43/6/78T was created in lambda zap express and screened using BJL/AC1. Single positive clones were isolated and excised into the phagemid vector pBK-CMV. Phagemid DNA was purified and a single clone was selected for sequencing. The size of spirochaetal DNA insert was determined by digestion with restriction endonucleases EcoRI and PstI as being approximately 2.6 kb. The nucleotide sequence of the gene encoding the protein with which the antibody reacted was determined by cycle sequencing. The insert contained an open reading frame of 285 nucleotides. Translation of the nucleotide sequence into amino acid (aa) residues showed a sequence of 275 aa. Comparison of this sequence with databases revealed homology to pyruvate oxidoreductases from various organisms found in the gastroinestinal tract. These included the pyruvate ferredoxin oxidoreductase (POR) alpha submit of Helicobacter pylori (38.8% identity in 250 aa), pyruvate-flavodoxin oxidoreductase of Escherichia coli (28.7% identify in 258 aa) and Giardia intestinalis (25.1% identity in 251 aa). A significant level of homology was also observed with hyperthermophilic bacteria such as the POR of Thermatoga maritima (38.6% in 254 aa) and the 2-ketovalerate-ferredoxin oxidoreductase of Pyrococcus furiosus (34% in 262 aa).

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

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

PCR detection of Brachyspira aalborgi and Brachyspira pilosicoli in human faeces.

Previously-developed PCR protocols specific for the 16S rRNA gene of the intestinal spirochaetes Brachyspira aalborgi and Brachyspira pilosicoli were adapted for the detection of these species in human faeces, following DNA extraction and purification using mini-prep columns. The limits of detection in seeded faeces for B. aalborgi and B. pilosicoli respectively were 2x10(2) and 7x10(3) cells per PCR reaction, equivalent to 5x10(4) and 1x10(5) cells per g of faeces. The PCR techniques were applied to faecal samples from two patients with histological evidence of intestinal spirochaetosis. In the first patient, in whom B. aalborgi had been identified by 16S rDNA PCR from colonic biopsies, a positive amplification for B. aalborgi only was obtained from the faeces. The organism could not be isolated from these faeces. In the second patient, both colonic biopsies and faeces were PCR positive for B. pilosicoli only, and B. pilosicoli was isolated from the faeces. These new faecal PCR protocols should be valuable for future studies on the epidemiology of intestinal spirochaete infections in human populations, particularly as it is not currently possible to isolate B. aalborgi from faeces.

Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons.

Multilocus enzyme electrophoresis (MEE) analysis and comparisons of nearly complete 16S rRNA gene sequences (1416 nucleotide positions) were used to evaluate phylogenetic relationships among Serpulina hyodysenteriae strain B78T, S. innocens strain B256T, Brachyspira aalborgi strain 513AT, and eight uncharacterised strains of swine, avian, and human intestinal spirochaetes. From MEE analysis, nine strains could be assigned to five groups containing other intestinal spirochaetes (genetic distances between groups = 0.6-0.9). Chicken spirochaete strain C1 and B. aalborgi 513AT represented unique electrophoretic types and formed their own MEE groups. Despite MEE differences, the 11 strains had highly similar (96.3-99.9%) 16S rRNA sequences. These findings point out limitations of both MEE analysis and 16S rRNA sequence comparisons when used as solitary techniques for classifying intestinal spirochaetes related to Brachyspira/Serpulina species.

Genetic characterisation of intestinal spirochaetes and their association with disease.

Multilocus enzyme electrophoresis was used to assess genetic relationships amongst 175 isolates of anaerobic intestinal spirochaetes, including 72 isolates from individuals living in different parts of the world, 102 from pigs and one from a dog. Amongst porcine isolates belonging to the genus Serpulina, a possible new species was identified. All but one of the isolates from man were clustered with the canine isolate and 59 porcine isolates in a distinct group that we have previously called "Anguillina coli". The human and animal spirochaetes in this group had four-to-six axial flagella and most were recovered from individuals with diarrhoea. They included a strain of the so-called "Serpulina jonesii", that was not a true serpulina. These 71 human isolates were distributed into 44 electrophoretic types and had a mean genetic diversity of 0.32. These were further divided into 26 clonal groups. Three of these clones also contained porcine isolates, one of which was strain P43/6/78, the agent of porcine intestinal spirochaetosis. Four of the clones contained human isolates from different sources. One included isolates from Western Australian Aboriginal children and from Italian adults, and the other three contained isolates from Western Australian Aboriginal children and from homosexual males in Sydney, New South Wales. There were no known connections between these human populations. The other spirochaete of human origin was Brachyspira aalborgi, which was distinct from isolates in the genera Serpulina and "Anguillina". Both B. aalborgi and "A. coli" have been associated with human cases of intestinal spirochaetosis.(ABSTRACT TRUNCATED AT 250 WORDS)