Summary
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. Others have questioned the clinical significance of colonisation by B. aalborgi, but we suggest that isolates of “A. coli” may be the cause of some clinical cases of intestinal spirochaetosis.

Genetic characterisation of intestinal spirochaetes and their association with disease

The intestinal anaerobic spirochetes Treponema hyodysenteriae B78T (T = type strain), B204, B169, and A-1, Treponema innocens B256T and 4/71, Treponema succinifaciens 6091T, and Treponema bryantii RUS-1T were compared by performing DNA-DNA reassociation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell proteins, restriction endonuclease analysis of DNA, and 16S rRNA sequence analysis. DNA-DNA relative reassociation experiments in which the S1 nuclease method was used showed that T. hyodysenteriae B78T and B204 had 93% sequence homology with each other and approximately 40% sequence homology with T. innocens B256T and 4/71. Both T. hyodysenteriae B78T and T. innocens B256T exhibited negligible levels of DNA homology ( 96%) in electrophoretic profiles among T. hyodysenteriae strains, moderate levels of similarity (43 to 49%) between T. hyodysenteriae and T. innocens, and no detectable similarity between the profiles of either T. hyodysenteriae or T. innocens and those of T. succinifaciens, T. bryantii, and Escherichia coli. Restriction endonuclease analysis of DNA was not useful in assessing genetic relationships since there was heterogeneity even between strains of T. hyodysenteriae. Partial 16S rRNA sequences of the intestinal spirochetes were determined by using a modified Sanger method and were compared in order to evaluate the phylogenetic relationships among these and other spirochetes. The 16S rRNA sequences of T. hyodysenteriae B78T, B204, and A-1 were nearly identical (99.8 to 99.9% base sequence similarity). T. innocens B256T and 4/71 were closely related to the T. hyodysenteriae strains (99.4 and 99.0% similarity). Strains of T. hyodysenteriae and T. innocens exhibited low levels of 16S rRNA similarity (average, 76.5%) with T. pallidum, Borrelia burgdorferi, and various other spirochetes. The results of our investigations indicate that T. hyodysenteriae and T. innocens are distinct but related species of spirochetes. T. hyodysenteriae and T. innocens are only distantly related to T. pallidum, the type species of the genus Treponema, and to other spirochetes. Consequently, we propose that the species T. hyodysenteriae and T. innocens be transferred to a new genus, Serpula, gen. nov.

Reclassification of Treponema hyodysenteriae and Treponema innocens in a new genus, Serpula gen. nov., as Serpula hyodysenteriae comb. nov. and Serpula innocens comb. nov.

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.

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

The porcine intestinal spirochaetes: identification of new genetic groups

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.

Development of a Duplex PCR Assay for Detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli in Pig Feces

Three liquid media that differ slightly in composition but not in the method of preparation were developed for the propagation of Treponema hyodysenteriae and Treponema innocens. The three media are unique in that all components are sterilized by autoclaving before use. These media supported better growth of T. hyodysenteriae than did previously used liquid media.

R. A. Kunkle

Papers

Autoclaved liquid medium for propagation of Treponema hyodysenteriae.

Autoclaved Liquid MediumforPropagation of Treponema hyodysenteriae