Preparation, characterisation and applications of monoclonal antibodies raised against outer envelope components of Serpulina hyodysenteriae and Serpulina pilosicoli

Lee, Bong Joo (1996) Preparation, characterisation and applications of monoclonal antibodies raised against outer envelope components of Serpulina hyodysenteriae and Serpulina pilosicoli. PhD thesis, Murdoch University.

Abstract

Serpulina hyodysenteriae and Serpulina pilosicoli are the aetiological agents of swine dysentery (SD) and porcine intestinal spirochaetosis (PIS), respectively. Both diseases are economically significant to pig production, and S. pilosicoli also infects human beings, poultry and dogs. The diagnosis of SD and PIS has remained difficult because both organisms are fastidious and difficult to isolate, and other non-pathogenic spirochaetes such as Serpulina innocens, which are common inhabitants of the swine colon, may be confused with the pathogenic species. Therefore the major aim of this thesis was to develop specific reagents that could be used for the detection and identification of the pathogenic species. To achieve this the cell surface of a range of intestinal spirochaetes was examined, and monoclonal antibodies (mAbs) specific for surface components of the two pathogenic species were prepared. It was also intended that knowledge acquired from the use of these mAbs might provide a basis for future development of vaccines against the agents.

Lipooligosaccharide (LOS) initially was examined and targeted for mAb production. However, LOS profiles of the various Serpulina spp. had similar banding patterns and this material showed extensive antigenic cross-reactivity within and between species. A mAb produced against the LOS of S. hyodysenteriae strain WA1 (serogroup B) was strain specific. Attempts to use a mAb against LOS as a specific marker for this species were therefore abandoned.

Outer membrane protein (OMR) profiles of the various spirochaetes then were examined, and were found to be more species-specific. OMRs therefore were used as immunising antigens and two mAbs, BJL/SH1, which was specific for S. hyodysenteriae, and BJL/AC1, which was specific for S. pilosicoli, were produced. In Western blot analysis, BJL/SH1 reacted with a 30 kDa component present in all of the S. hyodysenteriae strains tested, whilst BJL/AC1 reacted with a 29 kDa component present in all of the S. pilosicoli strains tested. Immunogold labelling indicated that both antigens were exposed on the surface of the outer membrane. Indirect immunofluorescence tests (IFT) and slide agglutination tests (SAT) were developed using these mAbs, and could be used to detect and identify the respective spirochaetes in faecal samples (IFT) and in pure culture (IFT, SAT). These reagents therefore should prove to be useful for detection and identification of the two pathogenic agents.

Even though neither mAb caused growth inhibition in vitro, an attempt was made to see whether specific immunisation with the 30 kDa antigen of S. hyodysenteriae induced immunity, and hence might be a useful candidate as a vaccine. To do this, a lambda ZAP II bacteriophage library of S. hyodysenteriae strain P18A DNA was prepared, and E. coli colonies expressing the recombinant 30 kDa antigen were detected using mAb BJL/SH1. Five plasmids containing the DNA inserts encoding the antigen were established in E. coli cells, and it was confirmed that these cells produced the 30 kDa antigen using mAb BJL/SH1 in Western blot analysis of cell envelope extracts from the E. coli strain. The E. coli cells then were inactivated and used to immunise mice, and the serum prepared was shown to recognise the 30 kDa protein antigen. Five pigs then were immunised with one of the inactivated E.coli recombinant strains, and these animals also developed antibody against the 30 kDa antigen. Following challenge with S. hyodysenteriae, four of five immunised pigs developed SD, thus indicating that antibody against the 30 kDa antigen did not provide protection.

Item Type:	Thesis (PhD)
Murdoch Affiliation(s):	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/53690

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