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The identification and molecular epidemiology of human intestinal spirochaetes, with particular reference to Brachyspira aalborgi

Mikosza, Andrew Stanislaw John (2002) The identification and molecular epidemiology of human intestinal spirochaetes, with particular reference to Brachyspira aalborgi. PhD thesis, Murdoch University.

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Abstract

Intestinal spirochaetosis in humans (HIS) is a condition defined by the presence of a layer of spirochaetes attach by one cell end to the colorectal epithelium. The clinical significance of HIS is uncertain, but it has been linked to chronic diarrhoea and other abdominal complaints. Two anaerobic intestinal spirochaete species have been associated with HIS, these being Brachyspira pilosicoli, and B. aalborgi. B. pilosicoli has been commonly identified in the faeces of people from developing countries (-30%). B. aalborgi, a slow growing and fastidious spirochaete, has previously been isolated only twice from patients in Scandinavia, although it has been detected in colonic biopsies obtained from nonhuman primates.

Due to the extreme difficulty in culturing B. aalborgi, and to a lesser extent, B. pilosicoli, diagnostic polymerase chain reaction (PCR) tests based upon the 16S rRNA and NADH oxidase {nox) genes were developed to detect B. aalborgi or B. pilosicoli, thus bypassing this problem. These tests were optimized for these species, and tested for specificity using Brachyspira and other bacterial species.

A method of extracting bacterial DMA from paraffin embedded tissues (PET) samples was developed. The PCR tests were then applied to 105 colorectal PET samples obtained from 60 human patients, and one non-human primate (stumpy tailed macaque) with histological evidence of intestinal spirochaetosis. These biopsies were obtained via pathologists primarily located in Australia, but also from Norway, France, the USA, Japan, French Guyana, and Hong Kong. For certain patients, multiple biopsies were obtained.

The application of PCR demonstrated that B. aalborgi rather than B. pilosicoli was the main spirochaete species involved in HIS in these patients. B. aalborgi alone was detected in the biopsies of 39 patients (65.0%), three were positive for B. pilosicoli alone (5.0%), two were positive for both species (3.3%), and biopsies from 16 patients (26.7%) and one macaque were negative for all PCRs.

The PCR tests were adapted for the detection of B. aalborgi and B. pilosicoli 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 103 and 104 cells per 0.1 g of faeces. The PCRs were applied to faecal samples from three patients with HIS. B. aalborgi was identified by PCR from colonic biopsies in two of the patients, as well as in their faeces. In the third patient, both colonic biopsies and faeces were PCR positive for B. pilosicoli only, and B. pilosicoli was isolated from the faeces.

The diagnostic PCRs specific for B. aalborgi and B. pilosicoli were applied to 182 DNA samples extracted from the faeces of Dutch patients with chronic diarrhoea. Two of these samples (1.1%) were positive using the B. aalborgi PCR and these PCR products were shown to be from B. aalborgi when sequenced. Neither B. aalborgi nor B. pilosicoli therefore appear to be commonly associated with chronic diarrhoea in patients from a western society.

The near complete sequencing of the 16S rRNA gene (1410 bp) was performed on 15 of the PET samples from which the spirochaete had been previously speciated by diagnostic PCR. The 16S rDNA sequences of five isolates of B. aalborgi anti seven isolates of B. pilosicoli were also included. The 16S rDNA sequences were used to divide B. aalborgi into two main groups, both distinct from B. pilosicoli. Thirteen unique B. aalborgi sequences were identified, confirming the existence of numerous strains of the species. Partial sequencing of the 16S rRNA gene (915 bp) was performed on a larger group of samples, including 10 further human fixed tissue samples and six samples from non-human primates. The analysis retained the overall population structure of B. aalborgi, and indicated that the sequences from non-human primates may be derived from one or more novel species.

Partial sequencing of the nox gene (413 bp) was also performed to further differentiate the strains, and to determine if multiple strains could be detected colonizing individual patients. Eleven distinct sequences were identified from 22 patients. No strain variation could be detected in multiple biopsies taken from any patient, however strain variation was detected in three biopsy samples as well as five of the faecal samples from non-human primates. The population structure of B. aalborgi tietiuceti from the partial nox sequences correlated well with that deduced from the 16S rDNA sequences.

Amplified DNAs (428 bp) were produced from a 16S rDNA PCR applied to DNA obtained from PET samples containing B. aalborgi and B. pilosicoli, as well as to cultured cells of the two species, and these were cloned into Escherichia coli and then sequenced. The number of polymorphisms in DNA extracted from PET as compared to DNA from cells did not significantly increase. Four point mutations were identified in a total of eight clones obtained from B. aalborgi 513AT cells, while in DNA extracted from PET, six point mutations which were closely related to B. aalborgi were detected in eight clones obtained from a biopsy from an HIS patient. Two point mutations were identified in eight clones obtained from B. pilosicoli WesB cells, while in DNA extracted from two PET samples that were positive for B. pilosicoli, only one polymorphism was identified in eight clones. With two exceptions from the B. aalborgi clones, all the point mutations appeared to be random. These results suggest that sequences obtained from fixed tissue do not produce significantly greater inaccuracies that may be introduced by DNA fragmentation and damage, and the main cause of inaccuracies in sequences derived in this way is likely to be the number of PCR cycles used.

Item Type: Thesis (PhD)
Murdoch Affiliation: Division of Veterinary and Biomedical Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: repository@murdoch.edu.au. Thank you.
Supervisor(s): Hampson, David and Penhale, William
URI: http://researchrepository.murdoch.edu.au/id/eprint/52233
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