Browsing by Author "Strydom, Marliz"

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    The ostrich mycoplasma Ms02 partial genome assembly, bioinformatic analysis and the development of three DNA vaccines
    (Stellenbosch : Stellenbosch University, 2013-03) Strydom, Marliz; Botes, Annelise; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The South African ostrich industry is under enormous threats due to diseases contracted by the ostriches. H5N2 virus (avian influenza) outbreaks the past two years have resulted in thousands of ostriches having to be culled. However, the more silent respiratory infectious agents of ostriches are the three ostrich-specific mycoplasmas. Named Ms01, Ms02, and Ms03, these three mycoplasmas are responsible for dramatic production losses each year, due to their intrusive nature and the fact that no vaccines are currently available to prevent mycoplasma infections in ostriches. The use of antibiotics does not eradicate the disease completely, but only alleviates symptoms. The ostrich industry commissioned investigations into the development of three specific vaccines using the relatively novel approach of DNA vaccination. The concept of DNA vaccine development is based on the availability of complete genome sequences of the pathogen against which the vaccine is to be developed. This is necessary in order to identify vaccine candidate genes through comparative genomic studies. The Ms02 genome has previously been sequenced, resulting in 28 large contiguous sequences. This thesis used the technique of Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR) to attempt assembly of these 28 contiguous sequences. The number was reduced to 14 large contiguous sequences, which were then subjected to repetitive sequence analysis and open reading frame analysis. Bioinformatic software was also used to predict the origin of replication. The extent of repeats in the Ms02 genome is illustrated, as well as the problems with genome assembly when dealing with repetitive-rich and A+T-rich genomes as those of mycoplasmas. Previous studies determined the mycoplasma oppA gene to be a good vaccine candidate gene, due to its cytadherent properties. This thesis describes the development of three DNA vaccines containing the Ms02 oppA gene, and a preliminary attempt to prove expression of one of these vaccines in a cell culture-based system. The DNA vaccine vectors pCI-neo, VR1012, and VR1020 were chosen for the vaccine development. The Ms02 oppA gene was also cloned into the prokaryotic expression vector pGEX-4T-1 in order to express the OppA protein for purification. The purified protein may be used in future serological tests in ostrich vaccination trials. In this study the protein was used to elicit anti-OppA rabbit antibodies, which were used to attempt detection of the pCI-neo-driven OppA protein expression in an MDA cell line in a transfection study. However, preliminary findings could not detect expression, but did indicate that the currently used colorimetric western blot technique may not be sensitive enough. It is suggested that different cell lines need to be investigated. Further optimisations are also required to decrease the observed non-specific binding.