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Detection, identification and subspecific differentiation of Serpulina hyodysenteriae and Serpulina pilosicoli

Atyeo, Roslyn Frances (1998) Detection, identification and subspecific differentiation of Serpulina hyodysenteriae and Serpulina pilosicoli. PhD thesis, Murdoch University.

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Abstract

Serpulina hyodysenteriae is the causative agent of swine dysentery (SD), and Serpulina pilosicoli is the aetiological agent of intestinal spirochaetosis (IS). Infections with these two intestinal spirochaetes cause major economic losses to piggeries through poor feedconversion, weight loss, and mortality. Control of these diseases is complicated by difficulties in diagnosis, and a lack of knowledge of the epidemiology of the infections. In this study, some practical DNA-based techniques to identify, detect and characterise these species were developed and evaluated.

Polymerase chain reaction (PCR) tests were developed to identify members of the genus Serpulina, including S. hyodysenteriae, S. pilosicoli, S. intermedia and collectively S. innocens and S. murdochii. These tests were optimised using up to 209 isolates of the various species in the genus Serpulina, and were found to be specific, although some problems were experienced with the test for S. intermedia when it was used on spirochaetes isolated from chickens.

A number of protocols to process faecal material prior to PCR testing were evaluated. The best method directly applied to faecal material was diatomaceous earth DNA extraction (DEX) which could routinely detect 106 S. hyodysenteriae cells in 0.2g of faeces. When a culture step was incorporated prior to DEX extraction of plate growth (CRDEX), approximately 1 o3. 104 S. hyodysenteriae cells or 104-105 S. pilosicoli cells in 0.2g of faeces were detected. Variation was observed with different faecal samples and different strains of spirochaetes used in seeding. The two techniques DEX and CRDEX also produced the best results when applied to faeces from experimentally or naturally infected pigs.

Pulsed-field gel electrophoresis (PFGE) was developed and evaluated as a molecular typing tool, and was successfully applied to 40 isolates of S. hyodysenteriae and 53 isolates of S. pilosicoli. The technique identified more genetic variation amongst isolates of S. pilosicoli (40 PFGE types) than amongst S. hyodysenteriae isolates (23 PFGE types). PFGE was a better means of differentiating S. hyodysenteriae isolates than were previous methods which have been used for subspecific differentiation, such as serotyping, DNA restriction endonuclease analysis and multilocus enzyme electrophoresis. Epidemiological studies, using PFGE, of isolates from nine farms naturally infected with spirochaetes showed more genetic variation amongst isolates of S. pilosicoli (12 PFGE types amongst 24 isolates from five farms) than for S. hyodysenteriae (seven PFGE types amongst 29 isolates from four farms).

An attempt was made to use PFGE to devise a physical map of the S. pilosicoli genome. The genome was estimated to be approximately 2.7Mb, and a partial physical map based on a small number of restriction digests was created.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary Studies
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
URI: http://researchrepository.murdoch.edu.au/id/eprint/53573
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