Browsing by Author "Jonker, Hester Isabella"

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    DNA vaccines against mycoplasma elicit humoral immune responses in ostriches
    (Frontiers Media, 2019-05-14) Wium, Martha; Jonker, Hester Isabella; Olivier, Adriaan Jacobus; Bellstedt, Dirk Uwe; Botes, Annelise; Leite, Luciana
    In ostriches, the population densities resulting from intensive rearing increases susceptibility to pathogens such as mycoplasmas. In addition to good management practices, vaccination offers an attractive alternative for controlling mycoplasma infections in food animals, instead of using antibiotics, which often leave unacceptable residues. The use of live attenuated vaccines, however, carry the concern of reversion to virulence or genetic recombination with field strains. Currently there are no commercially available vaccines against ostrich-infecting mycoplasmas and this study therefore set out to develop and evaluate the use of a DNA vaccine against mycoplasma infections in ostriches using an OppA protein as antigen. To this end, the oppA gene of “Mycoplasma nasistruthionis sp. nov.” str. Ms03 was cloned into two DNA vaccine expression vectors after codon correction by site-directed mutagenesis. Three-months-old ostriches were then vaccinated intramuscularly at different doses followed by a booster vaccination after 6 weeks. The ability of the DNA vaccines to elicit an anti-OppA antibody response was evaluated by ELISA using the recombinant OppA protein of Ms03 as coating antigen. A statistically significant anti-OppA antibody response could be detected after administration of a booster vaccination indicating that the OppA protein was successfully immunogenic. The responses were also both dose and vector dependent. In conclusion, the DNA vaccines were able to elicit an immune response in ostriches and can therefore be viewed as an option for the development of vaccines against mycoplasma infections.
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    Evaluation of DNA vaccines against Mycoplasma nasistruthionis sp. nov. str. Ms03 infections in ostriches and the production of IgA heavy chain proteins
    (2017-03) Jonker, Hester Isabella; Botes, Annelise; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Ostrich products have internationally become very popular with South Africa being the world leader in this industry. An increase in the demand for ostrich products has influenced production strategies by intensifying rearing conditions through the use of feedlot systems. Intensive rearing creates ideal conditions for the spread of pathogens such as mycoplasmas, which is associated with a respiratory disease syndrome amongst feedlot ostriches. The three ostrich-infecting mycoplasmas, Ms01, Ms02 and Ms03, together with other secondary pathogens result in reduced production. Since there are no vaccines available against these ostrich-infecting mycoplasmas, three DNA vaccines have been developed in this laboratory. Each vaccine consisted of a eukaryotic expression vector (pCI-neo, VR1020 or VR1012) containing the Ms03 oppA gene as antigen. The first objective of this study was to re-evaluate the anti-OppA immune response elicited by the pCI-neo and VR1020vaccines in ostriches. A vaccination trial was conducted and both vaccines were administered intramuscularly at 100, 600 and 1200 μg/ml doses followed by a booster vaccination. The anti-OppA immune response elicited by these vaccines in the ostriches was measured by means of the ELISA technique. All of the VR1020 vaccine doses as well as the 100 and 600 μg/ml doses of the pCI-neo vaccine were able to elicit a statistically significant anti-OppA immune response after administration of a booster vaccination. Since mycoplasmas target the respiratory system of ostriches a mucosally administered vaccine should also be considered. Opposed to the intramuscular route of vaccination, which results in humoral immunity represented predominantly by IgG, mucosal administration would result in mucosal immunity represented by IgA production. For the measurement of IgA production, the ELISA requires secondary anit-IgA antibodies. Although the whole antibody is typically used for the production of secondary antibodies, it is possible to use only the region representing the heavy chain constant region. This can then be produced as a recombinant protein that will allow an easy reproducible source for the production of secondary antibodies. The second objective of this study was therefore to evaluate the baculovirus-insect expression system for the production of the ostrich IgA heavy chain constant region (IgAH) protein, as this system will allow glycosylation of the protein product. The IgAH gene was inserted into the pAB-6xHis transfer plasmid and together with the ProFold-ER1 baculovirus used to co-transfect Sf9 insect cells for the production of ProFold-ER1_IgAH by means of homologous recombination. The IgAH protein was successfully expressed as confirmed by using HisProbe-HRP as well as previously produced rabbit anti-ostrich IgA antibodies during western blot analysis.