The ostrich mycoplasma Ms01 : the identification, isolation, and modification of the P100 vaccine candidate gene and immunity elicited by poultry mycoplasma vaccines

Pretorius, Benita (2009-03)

Thesis

The South African ostrich industry is currently being threatened by respiratory disease in feedlot ostriches with dramatic production losses. Three ostrich-specific mycoplasmas, Ms01, Ms02 and Ms03 were identified to be associated with respiratory disease in ostriches in South Africa. There is currently no registered mycoplasma vaccine available for use in ostriches. In order to prevent mycoplasma infections in South African ostriches, the ostrich industry has launched an investigation into possible strategies for vaccine development. This thesis describes different strategies for the establishment of immunity in ostriches against the ostrich-specific mycoplasmas. Firstly, the effectiveness of existing poultry mycoplasma vaccines to provide protection in ostriches against ostrich mycoplasma infections was tested. To this end, ostriches received primary and secondary vaccinations with poultry mycoplasma vaccines against Mycoplasma synoviae or Mycoplasma gallicepticum, respectively, after which protection against ostrich-specific mycoplasma was evaluated. Even though the specific identity of the ostrich-specific mycoplasmas (Ms01, Ms02, and/or Ms03) responsible for subsequent infection of immunized ostriches was not determined, it was concluded that poultry mycoplasma vaccines do not provide protection against these mycoplasma infections in ostriches. This appears to be the result of low levels of antibody crossreactivity between mycoplasmas, highlighting the necessity for the development of specific vaccines against each of the individual ostrich-specific mycoplasmas. Secondly, the development of a DNA vaccine against Ms01 was investigated. With the aim of developing an Ms01-specific DNA vaccine, the entire Ms01 genome was sequenced using GS20 sequencing technology. Bioinformatic searches were launched for the identification of an appropriate vaccine candidate gene in the Ms01 genome. The P100 gene, showing a high degree of homology with the P100 gene of the human pathogen M. hominis, was subsequently identified. After successful cloning, and modification of ten specific codons within the gene to correct for alternative codon usage, the modified P100 gene of Ms01 is now ready for insertion into a suitable DNA vaccine vector, for subsequent use as a DNA vaccine in ostriches.

Die Suid-Afrikaanse volstruisbedryf word huidiglik bedreig deur respiratoriese siektes in voerkraal volstruise wat aansienlike produksieverliese tot gevolg het. Drie volstruis-spesifieke mikoplasmas, Ms01, Ms02 en Ms03 is geïdentifiseer wat ‘n rol te speel in respiratoriese siektes in volstruise in Suid-Afrika. Daar is huidiglik geen geregistreerde mikoplasma entstof beskikbaar vir gebruik in volstruise nie. Ten einde mikoplasma infeksies in volstruise te voorkom, het die Suid-Afrikaanse volstruisbedryf ‘n ondersoek geloods na moontlike strategieë vir entstof ontwikkeling. Hierdie tesis handel oor benaderinge om immuniteit in volstuise teen die volstruis-spesifieke mikoplasmas te induseer. Eerstens is die effektiwiteit van bestaande pluimvee mikoplasma entstowwe getoets vir beskerming in volstruise teen volstruis-spesifieke mikoplasmas. Met dit ten doel, is volstruise twee maal met pluimvee entstowwe teen Mycoplasma synoviae of Mycoplasma gallisepticum onderskeidelik geënt, waarna die beskerming teen Ms01 geëvalueer is. Alhoewel die presiese identiteit van die volstruis-spesifieke mikoplasmas (Ms01, Ms02 en/of Ms03) verantwoordelik vir die daaropvolgende infeksies in geïmmuniseerde volstruise nie bepaal is nie, is dit gevind dat die toediening van pluimvee entstowwe nie beskerming gebied het teen hierdie mikoplasma infeksies in volstruise nie. Dit blyk die gevolg te wees van die lae vlakke van antiliggaam kruis-reaktiwiteit tussen mikoplasmas, en beklemtoon dat die ontwikkeling van spesifieke entstowwe vir elk van die volstruis-spesifieke mikoplasmas individueel uitgevoer sal moet word. Tweedens is die ontwikkeling van ‘n DNA entstof teen Ms01 ondersoek. Met die doel om ‘n Ms01- spesifieke DNA entstof te ontwikkel, is die volledige Ms01 genoomvolgorde bepaal deur gebruik te maak van “GS20” volgordebepalingtegnologie. Daarna is bioinformatika soektogte geloods vir die identifisering van ‘n geskikte entstof kandidaat geen in die Ms01 genoom. Die P100 geen, wat hoë homologie toon met die menslike patogeen M. hominis se P100 geen, is geïdentifiseer in Ms01. Na suksesvolle klonering, en die modifisering van tien spesifieke kodons in die geen, is die gemodifiseerde P100 geen van Ms01 nou geskik vir invoeging in ‘n geskikte DNA entstof vektor, vir daaropvolgende gebruik as DNA entstof in volstruise.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/101533
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