Application of a functional genomics approach to the identification of vaccine subunits and diagnostic antigens for use in the control of swine dysentery
Song, Yong (2007) Application of a functional genomics approach to the identification of vaccine subunits and diagnostic antigens for use in the control of swine dysentery. PhD thesis, Murdoch University.
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The intestinal spirochaete Brachyspira hyodysenteriae is the causative agent of swine dysentery (SD), a diarrhoeal disease of pigs which has significant economic impact worldwide. Controlling SD remains problematic, particularly as there is no effective vaccine and there are few definitive diagnostic methods available. In this study, a partial genomic sequence of B. hyodysenteriae was screened in silico. A total of 19 putative open reading frames (ORFs) encoding outer-membrane proteins then were selected and these were subjected to a laboratory screening process. To select potential universal vaccines, a preliminary study was conducted using PCR to determine the distribution of the putative genes in 23 strains of B. hyodysenteriae. A total of 17 of the 19 ORFs were considered to be suitable for further testing as they were found to be present in the majority of strains investigated. After molecular cloning and protein expression and purification, of 19 cloned candidate molecules derived from 17 genes (one large gene was divided into two parts encoding N and C terminal proteins, respectively), 14 were expressed in E. coli and the recombinant proteins were successfully produced. A variety of sera from pigs naturally and experimentally infected with B. hyodysenteriae were tested for reactivity with the 14 recombinant proteins in an immunoblotting assay. Seven molecules from six genes reacted strongly with the tested sera, and therefore were selected and used to immunize mice. All these proteins generated a specific antibody response. Post-immunization sera raised against each recombinant protein had the capacity to agglutinate B. hyodysenteriae cells, and also recognized the cognate proteins of B. hyodysenteriae in cell extracts. Further sequencing analysis demonstrated that these molecules were highly conserved in the genomes of different B.hyodysenteriae strains. Therefore, from the genomic based study, the products of six genes were identified as promising candidates for vaccines or as diagnostic targets.
Four genes were expressed on a large scale, the product (NAV-H7, NAV-H17 Cterminal, NAV-H34 and NAV-H42) were combined into one vaccine, and then this preparation was used to immunise pigs that subsequently were challenged with B. hyodysenteriae. These antigens generate systemic and colonic antibody responses, and vaccination tended both to delay the onset of clinical signs and attenuate lesion development. Hence these recombinant proteins showed promise as components for further SD vaccines. Recombinant proteins from the selected genes also were used as antigens in class-specific ELISAs used as serological assays for SD. Three antigens (NAV-H8, NAV-H42 and Bhlp29.7) were selected as good indicators of seroconversion in IgM ELISAs, and these were evaluated further using a large range of serum samples. The NAV-H8 IgM ELISA using a cut-off value 2.5 times the mean value of all negative pigs could be used as a herd test for SD, and both the NAV-H8 and NAV-H42 IgM ELISAs had potential for detecting exposure to B. hyodysenteriae at the pig level.
|Publication Type:||Thesis (PhD)|
|Murdoch Affiliation:||School of Veterinary and Biomedical Sciences|
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