2015-12-31 Preliminary investigations into the use of DNA vaccines to elicit protective immune responses against the ostrich mycoplasma Ms01

Brandt, Sonja (2012-03)

Thesis (MSc)--Stellenbosch University, 2012.

ENGLISH ABSTRACT: Mycoplasmas are evolutionarily advanced prokaryotes which have acquired the elite status of “next generation” bacterial pathogens and necessitate new paradigms in completely understanding their disease potential. The recurrent failure to eradicate mycoplasmal diseases from humans and animals through the use of antibiotics and other techniques have led to the conclusion that the most promising approach would be the development of an effective vaccine with which to control mycoplasma infections. The identification of three species of Mycoplasma which infect South African ostriches, causing huge losses to the South African ostrich industry each year, has thus prompted a search for new vaccination strategies with which to control and eradicate them. This study investigated the use of three potential DNA vaccines, utilizing the adherence-associated Ms01 OppA protein as antigenic determinant to generate antibody responses against the ostrich-infecting Ms01 organism. A vaccine trial in which the antigenic potential of the pCIneo, VR1012 and VR1020 DNA vaccines were evaluated in ostriches, necessitated the development of an enzyme-linked immunosorbent assay (ELISA) for serological analysis. To this end, the Ms01 oppA gene was isolated, cloned into a prokaryotic expression vector and expressed as a recombinant GST-fusion product in Escherichia coli. The successful expression and purification of the recombinant protein enabled its subsequent utilization as antigen in the generation of an ELISA. The ELISA displayed a high signal to background ratio. Using this ELISA, it could be shown that ostriches already possessed antibodies to the Ms01 organism prior to vaccination, a probable result of previous exposure. The expected antibody response pattern could not be detected in ostriches in response to the vaccinations, and therefore no final conclusion as to the immunostimulatory capabilities of the DNA vaccines could be drawn. Further vaccination trials in which ostriches that do not possess immunity to ostrich mycoplasmas, are required in order to obtain conclusive results.

AFRIKAANSE OPSOMMING: Mikoplasmas is evolusionêr gevorderde prokariote wat as nuwe generasie bakteriese patogene beskou word en nuwe paradigmas word benodig om hulle siekte potensiaal te verstaan. Die herhaaldelike mislukking om mikoplasmatiese siektes van mense en diere deur die gebruik van antibiotika en ander tegnieke het tot die gevolgtrekking gelei dat die mees belowende benadering tot die beheer van mikoplasma infeksies, die ontwikkeling van ‘n effektiewe entstof is. Die identifisering van drie spesies van Mycoplasma wat Suid- Afrikaanse volstruise infekteer, en groot verliese in die Suid-Afrikaanse volstruisbedryf veroorsaak, het tot ‘n soektog na nuwe entstof strategieë gelei waarmee hulle beheer en uitgewis kan word. Hierdie studie het die gebruik van drie potensiële DNA entstowwe ondersoek, wat die selaanhegtings-geassosieerde Ms01 OppA proteïen as antigeniese determinant benut, om antiliggaam response teen die volstruis infekterende Ms01 organisme te genereer. ‘n Entstof proef is onderneem waarin die antigeniese potensiaal van die pCIneo, VR1012 en VR1020 DNA entstowwe geëvalueer is in volstruise, en het die ontwikkeling van ‘n ELISA vir die serologiese analise daarvan genoodsaak. Vir die ontwikkeling van die ELISA, is die Ms01 oppA gene geïsoleer, gekloneer in ‘n prokariotiese ekspressie vektor en uitgedruk as ‘n rekombinante GST fusie produk in Escherichia coli. Die suksesvolle uitdrukking en suiwering van die rekombinante proteïen het die daaropvolgende gebruik daarvan as antigeen in die ontwikkeling van die ELISA moontlik gemaak. Die ELISA het ‘n groot sein tot agtergrond verhouding getoon. Die resultate wat met hierdie ELISA verkry is, het getoon dat die volstruise in hierdie inentingsproef blykbaar reeds voor immunisering antiliggame teen die Ms01 organisme besit het, wat op vorige kontak met die organisme dui. Die verwagte antiliggaam responspatroon kon dus nie in hierdie volstruise waargeneem word nie, en om dié rede kon geen finale afleiding oor die immuunstimulerende vermoëns van die DNA entstowwe gemaak word nie. Verdere inentingsproewe van volstruise wat nie vorige immuniteit teen volstruis mikoplasmas besit nie, word benodig om deurslaggewende resultate te verkry.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/20385
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