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Development of an efficacious recombinant vaccine for the obligate intracellular salmonid pathogen Piscirickettsia salmonis

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dc.contributor.author Kuzyk, Michael Allan
dc.date.accessioned 2018-02-21T19:10:28Z
dc.date.available 2018-02-21T19:10:28Z
dc.date.copyright 2000 en_US
dc.date.issued 2018-02-21
dc.identifier.uri https://dspace.library.uvic.ca//handle/1828/9087
dc.description.abstract Piscirickettsia salmonis is the aetiological agent of salmonid rickettsial septicaemia (SRS), an economically devastating rickettsial disease of farmed salmonids. SRS responds poorly to antibiotic treatment and no effective vaccine is available for its control. A molecular biology approach was used to characterize and identify antigens of P. salmonis that would be suitable to use as a recombinant subunit vaccine to aid in the control of SRS. A system for routine and reliable growth of P. salmonis was established using a chinook salmon (Oncorhynchus tshawytscha) embryo cell line. A purification protocol to separate P. salmonis from host cell material was devised using a combination of differential and Percoll density gradient centrifugation. Purified P. salmonis was used to generate polyclonal rabbit antisera. Indirect immunofluorescence microscopy, immunogold transmission electron microscopy, and biotin labeling of intact P. salmonis confirmed that P. salmonis was effectively separated from host cell debris and that immunoreactive antigens identified by rabbit antisera were surface associated. Rabbit anti- P. salmonis sera recognized the lipooligosaccharide component of bacterial lipopolysaccharide, and 7 protein antigens with relative mobilities of 27, 24, and 16 kDa and 4 migrating between 50–80 kDa. P. salmonis lipopolysaccharide was observed to be predominantly low m.w., but less abundant high m.w. species containing O-antigen were present. Genomic DNA was isolated from purified P. salmonis and used to construct an expression library in lambda ZAP II. In the absence of preexisting DNA sequence, rabbit polyclonal anti-P. salmonis serum was used to identify immunoreactive clones. A lambda clone encoding an immunoreactive 17 kDa outer surface protein (OspA) of P. salmonis was identified. The 4,983 by insert contained a high molar percentage of adenine and thymine, encoded four intact ORF's, and represented the first non-ribosomal DNA sequence data from P. salmonis. OspA is modified as a bacterial lipoprotein in Escherichia coli and is most closely homologous to a rickettsial 17 kDa surface lipoprotein previously only observed within the genus Rickettsia. A codon optimized version of ospA was constructed and the lipoprotein nature of OspA was determined to be a limiting factor in its production in E. coli. High level production of immunoreactive OspA targeted to inclusion bodies was achieved in E. coli by combining OspA with an N-terminal fusion protein. The OspA fusion was recognized by convalescent salmon sera thereby identifying OspA as an excellent candidate for a recombinant vaccine against P. salmonis. Vaccine preparations using P. salmonis bacterins were found to elicit variable immune responses in coho salmon (Oncorhynchus kisutch) that resulted in either protection or immunosuppression of vaccinates which varied with antigen dosage. Recombinantly produced OspA elicited an astonishing level of protection in vaccinated coho salmon with a relative percent survival (RPS) as high as 59%. In an effort to further improve the efficacy of the OspA recombinant vaccine, T cell epitopes (TCE's) from tetanus toxin and measles virus fusion protein which are universally immunogenic in mammalian immune systems were incorporated into an OspA fusion protein. Addition of the TCE's dramatically enhanced the efficacy of the OspA vaccine, reflected by a 3-fold increase in the number of coho salmon protected (83% RPS). These results represent an effective monovalent recombinant subunit vaccine for the rickettsial pathogen, P. salmonis. en_US
dc.language English eng
dc.language.iso en en_US
dc.rights Available to the World Wide Web en_US
dc.subject Rickettsial diseases en_US
dc.subject Rickettsial vaccines en_US
dc.title Development of an efficacious recombinant vaccine for the obligate intracellular salmonid pathogen Piscirickettsia salmonis en_US
dc.type Thesis en_US
dc.contributor.supervisor Kay, William Wayne
dc.degree.department Department of Biochemistry and Microbiology en_US
dc.degree.level Doctor of Philosophy Ph.D. en_US
dc.description.scholarlevel Graduate en_US


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