The development of a DNA vaccine against Mycoplasma nasistruthionis sp. nov. for use in ostriches

[摘要] ENGLISH ABSTRACT: Mycoplasma nasistruthionis sp. nov. str. Ms03 (Ms03) is one of three Mycoplasma speciesthat were identified from ostriches. Mycoplasmas infections have been implicated in ostrichchick mortalities, growth retardation and downgrading of ostrich carcasses. Currently there isno vaccine available for the treatment of mycoplasmosis in ostriches. This study investigatedthe development of DNA vaccines against Ms03 infections in ostriches. To this end, theMs03 genome was sequenced and annotated. The vaccine candidate gene, oppA, wasidentified within the genome sequence and characterized before DNA vaccines containingthe oppA were developed and tested. The genome of Ms03 was sequenced and the resulting 172 contigs were annotated. Thisdissertation presents the first Ms03 draft genome and annotation which contributed to theunderstanding of Ms03 as a miniature genetically independent organism. In Ms03, genomereplication, cell division, RNA transcription, protein translation and glycolysis resemble thatof the closely related Mycoplasma synoviae 53. Purine and pyrimidine metabolism wasincomplete and de novo synthesis thereof was not possible. Amino acid synthesis in Ms03was mostly absent and only the genes that convert aspartate to asparagine and glycine toserine were found. More importers than exporters were annotated owing to the lack ofsynthesis pathways in Ms03, which is typical for mycoplasmas that have parasitic life styles.Two oligopeptide permease (opp) operons were annotated within the Ms03 genome. The potential of the oppA as a vaccine candidate gene was evaluated by investigating theneed for a substrate-binding domain (OppA) as part of the OppBCDF transporter withinMycoplasma species. An oppA homologue could be identified for each oppBCDF operon inall species and therefore must play an essential role in oligopeptide transport. Allmycoplasmas (except for hemoplasma) had one, two or three opp operons that could bedivided into three types (Type A, B and C). Each type had unique InterPro and MEMEdomains and motifs which together with the phylogenetic analysis suggest unique roles intheir survival under different conditions. Ms03 had a Type A and a Type B opp operon, theType A oppA was used as vaccine candidate gene. The Type A oppA was cloned and site-directed mutagenesis was used for codon correctionbefore the mutated gene was sub-cloned into three DNA vaccine vectors. The three DNAvaccines (pCI-neo_oppA, VR1012_oppA and VR1020_oppA) were used to vaccinateostriches and the OppA-antibody response was analysed by ELISA. The VR1020_oppA andpCI-neo_oppA constructs elicited a primary immune response in ostriches, indicating thatthe OppA protein was expressed in vivo and was immunogenic. This can therefore beviewed as the first step in the development of a DNA vaccine for the control of mycoplasmainfections in ostriches.

[发布机构] Stellenbosch University