Detection by PCR and isolation assays of the anaerobic intestinal spirochete Brachyspira aalborgi from the feces of captive nonhuman primates

The purpose of this study was to investigate the presence of the anaerobic intestinal spirochetes Brachyspira aalborgi and Brachyspira pilosicoli in the feces of captive nonhuman primates (n = 35) from 19 species housed at the Zoological Gardens, Perth, Western Australia. Both spirochete species are known to infect human beings. DNA was extracted from freshly collected feces with a commercially available QIAamp DNA stool minikit and subjected to PCR protocols amplifying portions of the 16S rRNA genes of the two spirochete species. The feces were also subjected to selective culture for the spirochetes. Subsequently, feces from 62 other captive animals or birds representing 39 species at the zoo were examined by PCR to determine whether they were reservoirs of infection. Six fecal samples from individuals from four primate species (two vervet monkeys, two Tonkean macaques, one Japanese macaque, and one hamadryas baboon) tested positive in the B. aalborgi PCR. B. aalborgi was not detected by PCR in any of the other animal or bird species tested, and B. pilosicoli was not detected in the primates or any of the other animals or birds. B. aalborgi was isolated from both PCR-positive vervet monkeys. This is the first time that B. aalborgi has been isolated from nonhuman primates and the first time that it has been isolated from the feces of any species

Identification and recombinant expression of a 30 kDa outer envelope lipoprotein of Brachyspira hyodysenteriae

Swine dysentery (SD) is a contagious mucohaemorrhagic diarrhoeal disease of pigs, characterised by extensive inflammation and necrosis of the epithelial surface of the large intestine (1). Economic losses due to SD result mainly from growth retardation, costs of medication, and mortality (2). To date, no completely sensitive and specific assays are available for the detection of antibodies against the aetiological agent, Brachyspira hyodysenteriae. Recently, we identified and cloned a 30 kDa outer membrane lipoprotein (BmpB) which is specific to B. hyodysenteriae and is recognised by experimentally and naturally infected pigs. We propose the use of recombinant BmpB as an antigen for future development of an improved serological test for SD diagnosis

Analysis of genetic variation in Brachyspira aalborgi and related spirochaetes determined by partial sequencing of the 16S rRNA and NADH oxidase genes

The purpose of this study was to investigate genetic variation in the anaerobic intestinal spirochaete Brachyspira aalborgi by partial sequencing of the 16S rRNA and NADH oxidase genes. The spirochaete is poorly cultivable; hence, only six isolates were available for analysis. Additional sequences were amplified from DNA extracted from fixed colorectal biopsies from 26 patients with histological evidence of intestinal spirochaetosis, and from the faeces of six non-human primates (NHP). Multiple biopsies from sites along the large intestine were tested from three of the 26 patients. Sequences from two biopsies were closely related to those of the spirochaete Brachyspira pilosicoli. Eight B. aalborgi-like 16S rDNA sequences were generated from the biopsies from the other 24 patients, and four from the NHP faeces. The B. aalborgi 16S rDNA sequences were divided into three clusters, 1, 2 and 4, with individual sequence similarities to the type strain ranging from 97.49 to 100%. All human isolates of B. aalborgi were located in cluster 1, as was the sequence of the so-called 'Brachyspira ibaraki'. All four 16S rDNA sequences from the NHP faeces and the two NHP isolates of B. aalborgi were located in cluster 4, which was distinct. Cluster 4 may represent a novel Brachyspira species. Evidence for multiple strains of B. aalborgi or other Brachyspira species was found in biopsies from two patients. In the three individuals from whom multiple biopsies were amplified, the sequences at each intestinal site were the same, indicating the presence of one dominant strain

Evaluation of a 23S rDNA polymerase chain reaction assay for identification of Serpulina intermedia, and strain typing using pulsed-field gel electrophoresis

A polymerase chain reaction assay, amplifying a 1027 base pair portion of the 23S rDNA gene, was evaluated for identification of the intestinal spirochaete Serpulina intermedia. A total of 34 strains of S. intermedia isolated from pigs and chickens and 195 strains of other related species were tested. The optimised assay correctly identified all the S. intermedia strains, but generated 11 false positive reactions, giving a test sensitivity of 100% and a test specificity of 94.3%. The false positive reactions were generated from strains of four different species of intestinal spirochaetes, and the product was of the original predicted size. This suggests that the primer sites selected on the 23S rRNA gene were not completely specific for S. intermedia. Pulsed-field gel electrophoresis was then developed to investigate diversity amongst the S. intermedia strains. All strains tested had distinct DNA banding patterns using Mlu1, although three isolates from chickens on the same farm appeared closely related. The collection exhibited considerable genetic diversity, and strains from pigs and chickens were distributed in clusters throughout the dendrogram produced. The most closely related porcine and avian strains shared only 62% similarity

Population structure and molecular epidemiology of intestinal spirochaetes isolated from animals and man.

Serpulina hyodysenteriae and Serpulina pilosicoli are the respective antiological agents of swine dysentery and intestinal spirochaetosis. Both species have been shown to be genetically diverse but the mechanisms accounting for the variation have not been investigated. Originally, most bacterial species were thought to be clonal and although recombination occurred it was presumed to be too infrequent to disrupt linkage disequilibrium. The recent application of the index of association (Ia) to MEE data sets has shown that bacteria may occupy a spectrum of population structures. Whilst the majority of species are clonal, some naturally transformable species have been shown to be panmicitc (ie high levels of recombination have disrupted linkage disequilibrium) whilst others have an intermediate population structure. The Ia was applied to 231 S. hyodysenteriae isolates that were divided into 50 electrophoretic types (ETs), and the population structure was found to be epidemic (ie significant levels of background recombination occurring with a large number of isolates restricted to certain ETs that may have survival or virulence advantages). In contrast 145 S. pilosicoli isolates obtained from humans in the Highlands of PNG divided in 27 ETs had a completely panmictic population structure. The results may reflects the different epidemiological aspects of each disease. The potential mechanisms for recombination in each species require further investigation

Identification of the gene encoding BmpB, a 30 kDa outer envelope lipoprotein of Brachyspira (Serpulina) hyodysenteriae, and immunogenicity of recombinant BmpB in mice and pigs

A gene encoding a 30 kDa outer envelope protein of the intestinal spirochaete Brachyspira (Serpulina) hyodysenteriae, was cloned and expressed in Escherichia coli strain XLOLR. Five phagemids containing DNA inserts encoding the protein were established and one clone (pSHA) was sequenced. An 816 bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site (AGGAG), and putative -10 (TATAAT) and -35 (TTGAAA) promoter regions upstream from the ATG start of the ORF. A 12 bp inverted repeat sequence, possibly serving as a transcription terminator, was identified downstream from the TAA stop codon. Analysis of the amino acid sequence identified a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. Further analysis of the amino acid usage of this lipoprotein, designated BmpB, showed its possible outer membrane localisation. Comparison of the gene encoding the lipoprotein, bmpB, with GenBank nucleotide sequences showed that it has homology with the gene (plp3) encoding Plp3, an outer membrane lipoprotein of Pasteurella haemolytica (54% identity in 735 bp). Comparison of the deduced amino acid sequence with the SWISS-PROT amino acid database revealed greatest homology with the outer membrane lipoproteins (Plp1, 2, 3) of P. haemolytica (34% identity in 242 aa, 37% identity in 250 aa, and 39% identity in 272 aa, respectively), and lipoproteins (rcsF and lipoprotein-28) of E. coli (40% identity in 267 aa and 36% identity in 263 aa, respectively). Three of the recombinant E. coli clones (pSHA, pSHD, and pSHE) were formalinised and used to immunise mice. A bacterin preparation of one recombinant E. coli clone (pSHA) was used to immunise pigs. Sera from these mice and pigs recognised the 30 kDa lipoprotein in outer membrane preparations of B. hyodysenteriae, indicating the immunogenicity of recombinant BmpB. Sera from pigs naturally infected with B. hyodysenteriae also reacted with recombinant BmpB expressed in E. coli

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 amplification from fixed tissue indicates frequent involvement of Brachyspira aalborgi in human intestinal spirochetosis

PCR procedures amplifying portions of the 16S rRNA and NADH oxidase genes of Brachyspira aalborgi and Serpulina pilosicoli were applied to DNA extracted from paraffin-embedded human colonic or rectal tissues from 30 Norwegian, Australian, and U.S. patients, 16 of whom had histologic evidence of intestinal spirochetosis (IS). B. aalborgi-specific sequences were identified by PCR in 10 of the IS patients (62.5%) but none of the others, while S. pilosicoli sequences were not detected in tissues from any patient. Direct sequencing of products from three of the positive samples provided further confirmation of the presence of B. aalborgi. B. aalborgi may be a more common cause of intestinal spirochetosis than has been previously thought

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 2×102 and 7×103 cells per PCR reaction, equivalent to 5×104 and 1×105 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

Population genetic analysis of Serpulina pilosicoli and its molecular epidemiology in villages in the Eastern Highlands of Papua New Guinea

The population genetics of Serpulina pilosicoli and its molecular epidemiology in villages in the Eastern Highlands province of Papua New Guinea were investigated. Multilocus enzyme electrophoresis (MLEE) was used to analyse 164 isolates from humans and animals. These were divided into 33 electrophoretic types (ETs), four of which contained 65% of the isolates. The mean genetic diversity (n = number of ETs) for 145 human isolates was 0.18, and the mean number of alleles at five polymorphic loci was 2.6. The species appeared to be recombinant, as there was a lack of linkage disequilibrium, and 25% of all the possible combinations of alleles was present in the population. PFGE analysis using the enzymes MluI and Sall divided 157 of the isolates into 99 PFGE types, demonstrating the existence of considerable strain diversity in a geographically restricted area. The two techniques were in excellent agreement; however, PFGE was more discriminatory for strain typing than was MLEE. Nine out of 19 (474%) culture-positive individuals were colonized by the same PFGE type of S. pilosicoli when retested after 6 weeks. For three individuals, the PFGE profiles of the second isolate differed from the first in only one or two DNA bands, while the other seven individuals were colonized with distinct PFGE types on each occasion. In two cases, strains with the same PFGE pattern were isolated from humans and dogs, suggesting that cross- species transmission of S. pilosicoli may occur naturally and that the infection can be zoonotic