Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Mycoplasma nasistruthionis sp. nov. str. Ms03 (Ms03) is one of three Mycoplasma species that were identified from ostriches. Mycoplasmas infections have been implicated in ostrich chick mortalities, growth retardation and downgrading of ostrich carcasses. Currently there is no vaccine available for the treatment of mycoplasmosis in ostriches. This study investigated the development of DNA vaccines against Ms03 infections in ostriches. To this end, the Ms03 genome was sequenced and annotated. The vaccine candidate gene, oppA, was identified within the genome sequence and characterized before DNA vaccines containing the oppA were developed and tested. The genome of Ms03 was sequenced and the resulting 172 contigs were annotated. This dissertation presents the first Ms03 draft genome and annotation which contributed to the understanding of Ms03 as a miniature genetically independent organism. In Ms03, genome replication, cell division, RNA transcription, protein translation and glycolysis resemble that of the closely related Mycoplasma synoviae 53. Purine and pyrimidine metabolism was incomplete and de novo synthesis thereof was not possible. Amino acid synthesis in Ms03 was mostly absent and only the genes that convert aspartate to asparagine and glycine to serine were found. More importers than exporters were annotated owing to the lack of synthesis 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 the need for a substrate-binding domain (OppA) as part of the OppBCDF transporter within Mycoplasma species. An oppA homologue could be identified for each oppBCDF operon in all species and therefore must play an essential role in oligopeptide transport. All mycoplasmas (except for hemoplasma) had one, two or three opp operons that could be divided into three types (Type A, B and C). Each type had unique InterPro and MEME domains and motifs which together with the phylogenetic analysis suggest unique roles in their survival under different conditions. Ms03 had a Type A and a Type B opp operon, the Type A oppA was used as vaccine candidate gene. The Type A oppA was cloned and site-directed mutagenesis was used for codon correction before the mutated gene was sub-cloned into three DNA vaccine vectors. The three DNA vaccines (pCI-neo_oppA, VR1012_oppA and VR1020_oppA) were used to vaccinate ostriches and the OppA-antibody response was analysed by ELISA. The VR1020_oppA and pCI-neo_oppA constructs elicited a primary immune response in ostriches, indicating that the OppA protein was expressed in vivo and was immunogenic. This can therefore be viewed as the first step in the development of a DNA vaccine for the control of mycoplasma infections in ostriches. / AFRIKAANSE OPSOMMING: Mycoplasma nasistruthionis sp. nov. str. Ms03 (Ms03) is een van drie mikoplasma spesies wat volstruise infekteer. Mikoplasma-infeksies in volstruise veroorsaak kuiken vrektes, vertraagde groei en afgradering van volstruis karkasse. Daar is tans geen geregistreerde mikoplasma entstof beskikbaar vir gebruik in volstruise nie. Hierdie studie het die ontwikkeling van DNS-entstowwe teen Ms03-infeksies in volstruise ondersoek. Vir hierdie doel was die Ms03-genoomvolgorde bepaal en geannoteer. Die entstofkandidaat-geen, oppA, was geïdentifiseer in die genoomvolgorde en gekarakteriseer voordat DNS-entstowwe (wat die oppA-geen bevat) ontwikkel en getoets is. Die Ms03-genoomvolgorde was bepaal en die gegenereerde 172 aaneenlopende volgordes was geannoteer. Hierdie proefskrif bied die eerste voorlopige volgorde en annotering van die Ms03-genoom wat bygedra het tot die kennis van Ms03 as 'n miniatuur geneties onafhanklike organisme. Genoom-replikasie, seldeling, RNS-transkripsie, proteïen-translasie en glikolise in Ms03 stem ooreen met dié prosesse in die naverwante Mycoplasma synoviae 53. Die purien en pirimidien metabolisme was onvolledig en de novo sintese daarvan was nie moontlik in Ms03 nie. Aminosuursintese in Ms03 was meestal afwesig en net die gene wat aspartaat omskep na asparagien en glisien na serien was gevind in die annoteerde genoom. Meer invoerders as uitvoerders was geannoteer, wat dui op die gebrek aan sintesepadweë in Ms03. Dit is tipies van mikoplasmas wat ‘n parasitiese lewensstyle het. Twee oligopeptied-permeases (opp) operons was gevind in die Ms03-genoom. Die potensiaal van die oppA-geen as 'n entstofkandidaat-geen was geëvalueer deur die behoefte van 'n substraatbindingsdomein (OppA) as deel van die OppBCDF-vervoerder binne mikoplasma spesies te ondersoek. 'n Homoloog van die oppA-geen kon geïdentifiseer word vir elke oppBCDF-operon in al die spesies en behoort daarom 'n noodsaaklike rol te speel in die vervoer van oligopeptiede. Alle mikoplasmas (behalwe vir hemoplasmas) het een, twee of drie opp-operons, wat verdeel kan word in drie tipes (Tipe A, B en C). Elke tipe het unieke InterPro en MEME domeine en motiewe wat saam met die filogenetiese ontleding daarop dui dat hulle unieke rolle in oorlewing onder verskillende omstandighede speel. Ms03 het 'n Tipe A en Tipe B opp-operon, die Tipe A oppA is gebruik as entstofkandidaat-geen. Die Tipe A oppA-geen was gekloneer en teikengerigte-mutagenese was gebruik vir kodonregstellings voordat die gemuteerde geen in drie DNS-entstof vektore gesubkloneer was. Die drie DNS-entstowwe (pCI-neo_oppA, VR1012_oppA en VR1020_oppA) was gebruik om volstruise in te ent en die OppA-teenliggaamsreaksie was geanaliseer deur ELISA. Inenting met die VR1020_oppA en pCI-neo_oppA entstowwe het tot 'n primêre immuniteitsreaksie in volstruise gelei. Dit dui daarop dat die OppA proteïen in vivo uitgedruk en immunogenies was. Dit kan beskou word as die eerste stap in die ontwikkeling van 'n DNS-entstof vir die beheer van mikoplasma-infeksies in volstruise.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Ostrich farming is of significant economical importance in South Africa. Three ostrich mycoplasmas, Ms01, Ms02 and Ms03 have been identified previously, and were provisionally named ‘Mycoplasma struthiolus’ (Ms) after their host Struthio camelus. Ostrich mycoplasmas are the major causative organisms of respiratory diseases, and they cause stock losses, reduced production and hatchability, and downgrading of carcasses and therefore lead to large economic losses to the industry. In order to be pathogenic to their host, they need to attach through an attachment organelle, the so-called tip structure. This structure has been identified in the poultry mycoplasma, M. gallisepticum, and is made up of the adhesin GapA and adhesin-related CrmA. Currently, no ostrich mycoplasma vaccine is commercially available and for this reason the need to develop one has arisen. Therefore the first part of this study was dedicated to the identification and isolation of vaccine candidate genes in the three ostrich mycoplasmas. Four primer approaches for polymerase chain reactions (PCR’s), cloning and sequencing, were used for the identification of adhesin or adhesin-related genes from Ms01, Ms02 and Ms03. The primer approaches revealed that the target genes could not be identified due to the high diversity of sequences that were generated. Therefore sequences were also compared with those of other mycoplasma species in BLAST searches. Results showed that the most significant hit was with the human pathogen M. hominis oppD, which is located in the same operon as the membrane protein P100 involved in adhesion. Other hits were with ABC transporters which may also play a role in cytadhesion. The second part of this study was aimed at testing whether two poultry mycoplasma vaccines, M. synoviae and M. gallisepticum, can be used in ostriches to elicit immune responses until an ostrich mycoplasma vaccine has been developed. Ostriches on three farms of different age groups in the Oudsthoorn district were therefore vaccinated with these vaccines in a vaccine trial. The enzymelinked immunosorbent assay (ELISA) was used to test the level of antibody response. Results showed that both vaccines elicited an immune response in all three age groups. A high percentage of the ostriches reacted positively, which indicates that both vaccines elicit antibody responses and may therefore give protection against ostrich mycoplasma infections. / AFRIKAANSE OPSOMMING: Volstruisboerdery is ‘n belangrike ekonomiese sektor in Suid-Afrika. Drie volstruismikoplasmas, Ms01, Ms02 en Ms03, is voorheen geïdentifiseer en voorlopig ‘Mycoplasma struthiolus’ (Ms) benaam na aanleiding van hul gasheer, Struthio camelus. Volstruismikoplasmas is die grootste oorsaaklike organismes van respiratoriese siektes, kudde verliese en die afgradering van karkasse wat lei tot groot ekonomiese verliese in die volstruisbedryf. Ten einde patogenies vir die gasheer te wees, moet mikoplasmas deur middel van ‘n aanhegtingsmeganisme vasheg – die sogenaamde puntvormige struktuur. Hierdie struktuur is in die pluimvee mikoplasma M. gallisepticum geïdentifiseer, en bestaan uit aanhegting proteïen GapA en die aanhegting verwante proteïen CrmA. Tans is geen volstruismikoplasma entstof kommersieel beskikbaar nie, en derhalwe het die behoefte ontstaan om so ‘n entstof te ontwikkel. Die eerste gedeelte van hierdie studie is dus gewy aan die identifisering en isolering van entstof kandidaat gene in al drie volstruismikoplasmas. Vier inleier benaderings vir polimerase ketting reaksies (PKR), klonering asook geenopeenvolging bepalings vir die identifisering van aanhegting of aanhegting verwante gene vanuit Ms01, Ms02 en Ms03 is gebruik. Die inleier benaderings het getoon dat die teikengene nie geïdentifiseer kon word nie as gevolg van hoë variasie in die gegenereerde geenopeenvolgings. Derhalwe is geenopeenvolgings met ander mikoplasma spesies deur middel van BLAST soektogte vergelyk. Resultate het getoon dat die betekenisvolste ooreenstemming dié met die menslike patogeen M. hominis oppD was, wat deel vorm van die membraan proteïen P100 operon wat betrokke is by aanhegting. Ander ooreenstemmings sluit ABC transporters in wat moontlik betrokke kan wees by aanhegting. Die tweede gedeelte van hierdie studie het ten doel gehad om te toets of twee pluimvee mikoplasma entstowwe, M. synoviae en M. gallisepticum, gebruik kan word in volstruise om immuunresponse te ontlok tot tyd en wyl ‘n volstruismikoplasma entstof ontwikkel is. Volstruise vanaf drie plase in verskillende ouderdomsgroepe in die Oudtshoorn distrik was ingeënt met hierdie entstowwe in ‘n entstof proefneming. Die ensiem-afhanklike immuno-absorpsie essaï (ELISA) was gebruik om antiliggaam response te toets. Die resultate het getoon dat beide entstowwe immuunresponse ontlok het in al drie ouderdomsgroepe. ‘n Groot persentasie van die volstruise het positief gereageer wat ‘n aanduiding is dat beide entstowwe immuunresponse ontlok het en kan dus beskerming bied teen volstruismikoplasma infeksies.

Thesis (MSc)--Stellenbosch University, 2013. / 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. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse volstruisbedryf is onder geweldige druk vanweë siektes wat die volstruise bedreig. Die epidemie van die H5N2 virus (voëlgriep) in die afgelope twee jaar het veroorsaak dat duisende volstruise van kant gemaak moes word. Daar is egter nog ‘n bedreiging wat tot geweldige produksie verliese lei elke jaar: die respiratoriese infeksies wat versoorsaak word deur die drie volstruis mikoplasmas, genoem Ms01, Ms02 en Ms03. Geen entstowwe is tans beskibaar om die infeksies te voorkom nie, en behandeling met behulp van antibiotikas is nie effektief in die genesing van infeksie nie, maar help net om die simptome te verlig. Weens die erns van die saak, het die Suid-Afrikaanse volstruisbedryf ‘n ondersoek geloods na die ontwikkeling van enstowwe teen elkeen van die drie volstruis mikoplasmas. Die relatiewe nuwe benadering van DNA-entstof ontwikkeling was die strategiese keuse. Die beginsel van DNA-entstof ontwikkeling berus op die beskikbaarheid van die genoomvolgordes van die siekte-veroorsakende organisme waarteen die enstof ontwikkel word. Geskikte kandidaat entstof gene word so opgespoor met behulp van vergelykende studies met ander beskikbare genome. Die Ms02 genoomvolgorde is voorheen bepaal en word verteenwoordig deur 28 groot geenvolgorde fragmente. Die tegniek van Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR) is gebruik om van die 28 fragmente aan mekaar te las. Die aantal fragmente is verminder na 14 groot geenvolgorde fragmente, wat vervolgens gebruik was om die omvang van herhalende volgordes in die genoom te bepaal, om nuwe leesrame te ondersoek, asook om die oorsprong van DNA replikasie op te spoor met behulp van bioinformatika sagteware. Die omvang van die herhalende aard van die Ms02 genoom word geïllustreer, asook die gepaardgaande probleme met die las van geenvolgorde fragmente wanneer met genome van veelvuldige herhalende volgordes, wat boonop A+T-ryk is, gewerk word, soos die van mikoplasmas. Vorige studies het die mikoplasma oppA geen geïdentifiseer as ‘n geskikte kandidaat entstof geen as gevolg van sy selaanhegting-eienskappe. Hierdie studie behels die invoeging van die Ms02 oppA geen in drie DNA-enstof vektore, naamlik pCI-neo, VR1012, en VR1020, asook die voorlopige poging om bewys van uitdrukking van een van die entstowwe in ‘n selkultuursisteem te bewerkstellig. Die geen is ook gekloneer in die prokariotiese ekspressie vektor pGEX-4T-1, ten einde die Ms02 OppA proteïen te isoleer. Die geïsoleerde proteïen kan in serologiese toetse in toekomstige volstruis enstof proewe gebruik word. In hierdie studie is die proteïen gebruik om konyn teenliggame teen dit op te wek, wat dan gebruik was om vir die pCI-neo-gedrewe ekspressie van die oppA geen te toets in ‘n selkultuur omgewing deur ‘n MDA sellyn te transfekteer. Die voorlopige resultate toon nie ekspressie van die OppA proteïen aan nie, maar het wel uitgelig dat die western blot tegniek wat tans gebruik word, dalk nie sensitief genoeg is nie. Dit kan belowend wees om ander tipes selle te toets. Verdere optimisering is ook nodig om die nie-spesifieke binding wat waargeneem is, te verlaag. / South African Ostrich Business Chamber

Dissertation (PhD)--University of Stellenbosch, 2004 / ENGLISH ABSTRACT: Newcastle disease (NO) and mycoplasma infections in ostriches have considerable economic implications for the South African ostrich industry in that NO is a limiting factor in the export of ostrich products to the European Union and mycoplasma infections cause stock losses, reduced production, reduced hatchability and downgrading of carcasses. In the first section of this dissertation, the role of passively acquired and mucosal immunity in protection of ostrich chicks against Newcastle disease virus (NOV) was investigated. Ostrich hen serum IgG and yolk IgY were isolated and characterized, and the transfer of maternal anti-NOV antibodies to the egg yolk was determined using an enzyme-linked immunosorbent assay (ELISA). Results indicated that anti-NOV antibodies were successfully transferred from the ostrich hen to the egg yolk. In addition, ostrich IgA was isolated, characterized and rabbit anti-ostrich IgA antibodies produced and used for measuring mucosal anti- NOV IgA antibodies produced in response to mucosal vaccination. Results indicated that the live La Sota vaccine stimulates IgA production and thus mucosal immunity in ostrich chicks. In the second section of this dissertation, ostrich mycoplasmas were isolated and identified using 16S rRNA gene sequencing. These sequences indicated that ostriches carry three unique mycoplasmas, which are phylogenetically quite divergent. The 16S rRNA gene sequences of the ostrich mycoplasmas were subsequently used for the development of specific primers for the detection and diagnosis of mycoplasma infections in ostriches by PCR. The last section of this dissertation focuses on avian malaria in African penguins and the management of this disease during rehabilitation. The Foundation for the Conservation of Coastal Birds (SANCCOB) is a seabird rescue and rehabilitation centre, which is largely dedicated to the rehabilitation of diseased, injured and oiled penguins. Significant mortalities due to avian malaria occur at this facility. The aim of this study was the development of an ELISA for the purpose of assessing the natural levels of anti-Plasmodium antibodies in African penguins on entry into the SANCCOB facility and during rehabilitation. Results indicated significant increases in anti- Plasmodium antibody levels after entry, which was not influenced by oiling. Infection with malaria and not parasite recrudescence was viewed to be the cause of this increase, indicating a possible role of the SANCCOB facility in exposing penguins to avian malaria. / AFRIKAANSE OPSOMMING: Newcastlesiekte (NS) en mikoplasmainfeksies in voltruise het geweldige ekonomiese implikasies vir die Suid-Afrikaanse volstruisbedryf. Die rede hiervoor is dat NS 'n beperkende faktor in die uitvoer van volstruisprodukte na die Europese Unie is, en mikoplasmainfeksies tot kudde verliese, verlaagde produksie en uitbroei asook lae gradering van karkasse lei. In die eerste gedeelte van hierdie proefskrif is die rol van passiewe- en mukosale-immuniteit in die beskerming van volstruiskuikens teen NS virus (NSV) ondersoek. Volstruishenserum IgG en eier IgY is geïsoleer en gekarakteriseer en die oordrag van maternale anti-NSV antiliggame na die eier ondersoek met behulp van 'n 'enzyme-linked immunosorbent assay' (ELISA). Resultate het getoon dat anti-NSV antiliggame suksesvol van die hen na die eier oorgedra is. Volstruis IgA is ook geïsoleer, gekarateriseer en konyn anti-volstruis IgA antiliggame geproduseer wat gebruik is vir die bepaling van mukosale anti-NSV IgA antiliggame in reaksie op mukosale immunisering. Resultate het getoon dat lewendige La Sota entstof IgA produksie stimuleer en dus tot mukosale-immuniteit in volstruiskuikens lei. In die tweede gedeelte van hierdie proefskrif is volstruismikoplasmas geïsoleer en geïdentifiseer met behulp van 16S rRNA geenopeenvolgingsbepalings. Hierdie volgordes het getoon dat drie unieke mikoplasmas in volstruise voorkom wat filogeneties verskillend blyk te wees. Die 16S rRNA geenopeenvolgings van die volstruismikoplasmas is gebruik vir die ontwikkeling van spesifieke inleiers vir die PKR identifisering en diagnose van mikoplasmainfeksies in volstruise. Die laaste gedeelte van hierdie proefskrif fokus op voëlmalaria in die Afrika pikkewyn en die bestuur van hierdie siekte gedurende rehabilitasie. Die 'South African Foundation for the Conservation of Coastal Birds' (SANCCOB) is 'n seevoëlreddingsen rehabilitasie-sentrum vir siek, beseerde en ge-oliede pikkewyne. Hierdie sentrum het egter aansienlike vrektes as gevolg van voëlmalaria. In hierdie studie is 'n ELISA ontwikkel vir die bepaling van natuurlike anti-Plasmodium antiliggaamvlakke van pikkewyne by aankoms en tydens rehabilitasie by SANCCOB. Resultate het 'n toename in anti-Plasmodium antiliggaamvlakke getoon na toelating wat nie beïnvloed is deur olie nie. Hierdie toename kan toegeskryf word aan nuwe malariainfeksies en nie 'n heruitbraak van bestaande infeksies nie wat daarop dui dat pikkewyne aan voëlmalaria blootgestel word by die SANCCOB-sentrum.