Thesis (MScAgric (Animal Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: A study was conducted to characterize the three ostrich breeds available as genetic resource in South Africa, namely the South African Black (SAB), Zimbabwean Blue (ZB) and the Kenyan Redneck (KR), and their respective crosses. Growth, slaughter traits and reproduction of these ostriches were recorded at Oudtshoorn Research Farm in the Western Cape of South Africa. Individual non-linear regressions (Gompertz) were fitted to the data of 390 purebred and 41 crossbred ostriches, using the SAS NLIN function. Heterosis was estimated for each parameter of the Gompertz model. The estimated adult weight (Aparameter) of the ZB (147 kg) and the KR breeds (148 kg) were higher than that of the SAB breed (129 kg). The overall growth rate (B-parameter) of the ZB breed (0.0075) and the SAB breed (0.0080) was lower than that of the KR (0.0150). The age at maximum weight gain (C-parameter) was higher for the ZB breed (226 days) compared to the SAB (198 days) and the KR (194 days). Heterosis for the A-parameter was estimated at -6.2% and at -12% for the C-parameter. The slaughter traits studied were slaughter weight (SLW), carcass weight (CW), dressing percentage (DP), fan fillet weight (FFW), pH0, pH24, drip loss % (DL%), cooking loss % (CL%), tenderness and meat colour traits. Differences were observed between the means for SLW of the SAB (86.5 kg) and ZB (93.9 kg). Mean DP of the KR breed (52.5%) was increased relative to the low DP of their SAB contemporaries (48.8%). The sire lines (ZB and KR) and crosses were heavier than the SAB (dam line), whereas the crosses resembled the dam line for meat quality traits. Means for pH24 also differed, with higher values for the sire lines (ZB – 6.36; KR – 6.4) relative to the SAB (5.85). The instrumental b* colour value also differed between the SAB (9.4) and KR (6.9). Records used for assessing the reproduction and body measurements of purebred and crossbred dams were 428 in total. Traits analyzed were, total egg production (TEP), the number of fertile eggs, number dead in shell chicks, hatchability and chick production (CP), the time to lay, live weight, front chest circumferences as well as tail circumference. The ZB and KR were heavier in live weight and of larger body measurements than the SAB, whereas the SAB exhibited superior reproduction performance in comparison with the ZB and KR breeds. Derived heterosis estimates amounted to 2.2% for tail circumference, 12% for TEP, 12% for hatchability and 19% for CP. Genetic variation between and within the breeds were determined utilizing 19 microsatellite markers. Significant molecular genetic differences were observed between the three breeds. The SAB and ZB (Fst = 0.10 and Nei = 0.49) were genetically most similar, whereas the genetic distance between the KR and ZB breeds were furthest (Fst = 0.13 and Nei = 0.61). The SAB breed exhibited the highest heterozygosity within its population and the ZB the lowest heterozygosity. These results contribute to a better understanding of the utilization of the distinct ostrich breeds for commercial production. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die verskille tussen drie volstruisrasse wat tans in Suid Afrika mee geboer word te karakteriseer, naamlik die “South African Black” (SAB), “Zimbabwean Blue” (ZB) en die “Kenyan Redneck” (KR) en hulle onderskeie kruisse. Rekords van die groei-, slag- en reproduksie eienskappe van die volstruise was by Oudtshoorn Navorsingsplaas in die Wes-Kaap aangeteken. Individuele nie-lineêre regressies (Gompertz) is op die data van 390 suiwerras en 42 kruisgeteelde volstruise gepas, met die gebruik van die “NLIN” prosedure van SAS, (2006). Heterose is beraam vir elke parameter van die Gompertz model. Die beraamde volwasse gewig (A-parameter) van die ZB (147 kg) en die KR ras (148 kg) was hoër as die van die SAB ras (129 kg). Die totale groeitempo (B-parameter) van die ZB ras (0.0075) en die SAB ras (0.0080) was laer as die van die KR (0.0150). Die ouderdom by maksimum groei (C-parameter) was hoër vir die ZB ras (226 dae) in vergelyking met die SAB (198 dae) en die KR (194 dae). Heterose vir die A-parameter was beraam teen -6.2% en teen -12% vir die C-parameter. Die slageienskappe wat ondersoek was, was slagmassa (SLW), karkasmassa (CW), uitslag persentasie (DP), “fan fillet” massa (FFW), pH0, pH24, drupverlies % (DL%), kookverlies % (CL%), sagtheid en kleureienskappe. Beduidendende verskille is waargeneem tussen die gemiddeldes vir SLW vir die SAB (86.5 kg) en ZB (93.9 kg). Gemiddelde DP van die KR ras (52.5%) was beter as die van die SAB ras (48.8%). Die mannetjielyne (ZB en KR) en die kruisse was swaarder as die SAB (wyfielyn), en die kruise was vergelykbaar met die wyfielyn vir vleiskwaliteit eienskappe. Gemiddeldes vir die pH24 het verskil, met hoër waardes vir die vaar lyne (ZB – 6.36; KR – 6.4) relatief tot die SAB (5.85). Die instrumentale b* kleurwaarde het ook verskil tussen die SAB (9.4) en KR (6.9). ‘n Totaal van 428 rekords is gebruik om reproduksie en liggaamsmetings van die suiwer en kruisteelwyfies te ondersoek. Reproduksie eienskappe ge-analiseer was: die aantal broeisels, totale eierproduksie (TEP), die aantal vrugbare eiers, die aantal kuikens dood in dop, uitbroeibaarheid, kuiken produksie (CP), tyd tot produksie van die eerste eier, volwasse gewig, voorbors omtrek, sowel as, kruisomtrek. Die ZB en KR rasse was swaarder as die SAB, en het groter liggaamsmetings gehad. Die SAB het beter reproduksie in vergelyking met die ZB- en KR rasse gehad. Heterose beramings was 2.2% vir kruisomtrek, 12% vir TEP, 12% vir uitbroeibaarheid en 19% vir CP. Genetiese variasie tussen en binne die rasse was vasgestel deur die gebruik van 19 mikrosatelliete merkers. Beduidende genetiese verskille op ‘n molekulêre vlak was waargeneem tussen die drie rasse. Die SAB en ZB (Fst = 0.10 en Nei = 0.49) was geneties meer gelyk terwyl die KR en ZB genetiese verder verwyder is (Fst = 0.13 en Nei = 0.61). Die SAB ras het die hoogste heterosigositeit binne populasie getoon, en die ZB die laagste. Hierdie resultate dra by tot ‘n beter begrip van die gebruik van die drie rasse in kommersïele produksie.

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.

Thesis (PhD(Agric) Animal Sciences))--University of Stellenbosch, 1988. / Farming with ostriches became established as a new stock-farming activity in South Africa around 1863. Ostrich feathers were then the only commercial product of that activity and fifty years later still held fourth place on the list of exports from South Africa - after gold, diamonds and wool (Smit, 1963). However during the world depression of 1914 - 1945 the appeal of ostrich feathers vanished and the industry collapsed. Today commercial farming with domesticated ostriches (Struthi0 camelus var. domesticus), originating from a cross (Duerden, 1910; Smit, 1963, 1984) between the South African (Struthio camelus australis) and the North African ostriches (Struthio camelus camel us), is again a rapidly expanding activity in South Africa's Little Karoo. This is the only region in the world where farming with ostriches is still encountered at a commercial scale, and although relatively small in the general stock-farming scenario, it provides a livelihood for about 400 farmers who run some 150 000 ostriches. Feathers, together with ostrich leather and meat are all prominent export products that account for foreign exchange earnings of more than R30 million. In terms of monetary value the ostrich industry has grown by more than 300% over the past decade and by al most 100% in the 1ast 5 years. This high growth rate is primarily due to the steady development of the slaughter bird industry in which leather, meat and feathers account for more than 80% of the entire industry's turnover. Ostrich products, however, are primarily exclusive fashion products, of which more than 90% is exported. Because the world market is relatively small, expansion prospects for the industry are limited and sensitive to supply and demand. To ensure the industry's continued economic well-being, ostrich research pays particular attention to production techniques that will help to improve efficiency and result in better product quality and profitability. For the purpose of increased ostrich chick production, ostrich eggs are presently being incubated in large quantities (ca 200 000 eggs per annum). However, hatching success of ostrich eggs in incubators is considerably below that of natural nests, and Burger & Bertram (1981) suggested that it may be due to high humidities (63% relative humidity) commonly used in ostrich incubators. Investigations were undertaken to measure incubation temperatures and humidities during the complete course of 41 days of natural incubation in 6 ostrich nests. In addition, the water vapor conductance of the eggs was measured, as well as the incubation water loss which in other species averages 15% of the initial egg mass and has been proposed as an optimal condition for hatching success (Ar & Rahn, 1978; Rahn 1984). The natural incubation parameters measured during these experiments were adapted and applied to conditions of artificial incubation. This improved the realized hatchability from 50 to 75%. Furthermore the intensive production and finishing of slaughter birds on complete dry meal diets under feedlot conditions is a new development which contains exceptional possibilities for the industry (Swart & Kemm, 1985). Economically and biologically, efficient diet formulation has accordingly become essential for profitable slaughter bird production. The nutritional value of feed constituents for ostriches is, however, still unknown and without it effective least cost diet formulation and programming is not possible. Present growth and finishing diets for ostriches are based on energy values of the dietary components which have been derived from poultry (Blair, Daghir, Morimoto, Peter & Taylor, 1983; Du Preez, Duckitt & Paulse, 1986) and pig values (Kemm & Ras, 1981; NRC, 1973; IAFMM, 1985). This approach may, however, result in unreal estimation of the actual nutritional value of feed constituents for ostriches, so that quantification of their nutritional value has consequently become necessary. During 1985 a multi-disciplinary research programme on ostriches was initiated, the objective being to investigate the energy metabolism and the efficiency of energy utilization in growing farm ostriches. For the purpose of these experiments all ostrich chicks were incubated in an ostrich egg incubator, maintained at a dry bulb temperature of 36°C and a wet bulb temperature of 24,0 ± 1,0°C. These temperature settings were extrapolated from the natural incubation parameters measured in the six naturally incubated nests (Swart, Rahn & De Kock, 1987; Swart & Rahn, 1987). An important aspect of the studies on energy metabolism was to determine the extent to which plant fibre is digested in various segments of the gastrointestinal tract of growing ostriches and whether these birds utilize the end products of fibre digestion, namely volatile fatty acids such as acetic acid, efficiently. The possible use of metabolizable energy to evaluate feedstuffs for ostriches was an initial aim of this study. Subsequently the effect of crude fibre or energy concentration on the digestibility of gross energy, energy loss as methane, heat expenditure, and the effect on the efficiency of ME utilization were investigated. Finally accretion patterns and the partition of metabolizable energy during growth were studied in the young ostrich chick. The findings of the studies on incubation aspects (Section 1) are presented in Chapter 2 and 3 and that of the metabolism studies (Section 2) are presented in Chapters 4, 5, 6, 7 and 8 of this dissertation.

Thesis (PhD(Agric) (Animal Sciences))--Univsersity of Stellenbosch, 1992. / The anatomy of the muscles of the hindquarters of the ostrich is described and presented graphically for the purpose of the present and future studies in meat science involving the ostrich. In the past, the lack of an accurate anatomical description of this species has led to the incorrect identification of certain muscles by researchers. The process of growth is described for 51 components of the ostrich body, which includes 21 muscles and four organs. Gompertz functions were calculated to formulate growth models for these components. Acceptable goodness-of-fit was obtained for the formulated models regarding the raw skin area, mass of the head, heart, filet and other muscles as well as certain bones. No information on similar models could he found for the description of growth of individual muscles or body parts of other meat animals. However, similar growth models are available for the description of live mass changes over time. Since the general growth curve is sigmoidal it is difficult to fit accurate curves by arithmetical methods. This has led to the development of simpler linear models, which express the growth of a body part relative to the growth of the same entire body. Time based predictions of yield are thus impossible. In fact, many of these allometric equations are simply in a different mathematical form, rather than being totally different equations. Furthermore, the latter equations cannot be used to predict accurate values at the positive and negative extremes since unrealistic values are obtained. Computer programs are presented in the dissertation which can be used without modification on various species, such as fish, poultry, plants and forests, as well as micro organisms in plant pathology, food science and sewage. With the development in computer technology and by means of the suggested programs, it is possible to describe the growth process on a time scale with ease. It is also possible to estimate the mass of any body part at birth (and possibly at conception) as well as at maturity. Evidence is provided that unrealistic asymptotic values are obtained with time based linear and polynomial functions as well as with published non linear allometric models for the description of growth in the ostrich. A goodness-of-fit test for the Gompertz function is presented. It is advisable to carry out this test before fitting alternative models, since the relationship between two variables is only linear on a double log scale when both variables can be described by similar functions on a time scale. A method for the calculation of the standard error at the point where maximum growth occurs is also presented. These methods can successfully be used for any of the above mentioned species. The point at which maximum growth occurred was used to compare the muscles of the hindquarters of the ostrich with that of Merino rams. This showed that these two species differ markedly in this regard. Furthermore, it was proved that certain economically important muscles still gained weight after the optimal skin size (120 dm2) was reached at the age of ten months. It is also shown that the fat content of ostrich meat (filet) varied independently of the abdominal fat content, and that it reached a minimum value of 0.3% at the popular slaughter age of 14 months. Reliable methods for age estimation of a slaughtered bird is presented, based on the growth models. This information can be used to develop a classification system for ostrich meat and other meat animals.

Ostrich (Struthio camelus) breeding is a well-developed industry in South Africa. However, successful genetic management has yet to be implemented. Parentage in colony breeding ostriches is unknown, where for a given offspring, a number of possible parents exist. Molecular markers have been extensively used in the livestock industry to resolve parentage issues and are only beginning to be utilized to address the issues of the ostrich industry. The aims of this investigation were to test known microsatellite markers developed for other ostrich subspecies in a South African Black ostrich population, and to further test these markers for their use in individual and parentage identification. DNA was extracted from venous blood obtained from two pair bred families and a colony of 97 individuals. Eleven polymorphic microsatellite markers were tested by PCR amplification of DNA samples followed by multiplexing on polyacrylamide gels to generate DNA fingerprints for each individual. Alleles were sized and quantified and used to create genotypes for each individual. Parentage analysis was performed using exclusion and likelihood methods. Pedigrees were constructed for the families by comparison of genotypes. Breeding statistics were calculated for the colony individuals. Three microsatellite markers did not amplify in this population and one marker was found to be monomorphic in this population. Four of the microsatellite markers that successfully amplified produced anonymous amplification products suggesting a second annealing site in the genome sequence of Blacks. All loci displayed low observed heterozygosities indicative of little genetic variation in this population. For the colony sample, four individuals were not assigned either parent and one female did not contribute any offspring. On average females produced 4.86 ± 2.71 fertile eggs during the sampling period with a coefficient of variation of 55.86%. A total of 79.2% of individuals were assigned paternity and 88.3% were assigned maternity. A greater number of loci are required to improve the power of parentage analysis within breeding flocks incorporating all eggs laid. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Thesis (MScAgric (Animal Sciences))--University of Stellenbosch, 1985. / The domestication of ostriches and commercial ostrich breeding has a long and interesting history. Orstriches were successfully domesticated in Algeria during 1857 and also successfully bred in Italy during 1859. In South Africa the domestication of wild ostriches started in the Cape Colony with 80 tame birds during 1865. This number increased to 32 247 birds within ten years (De Mosenthal, 1877). Ostrich farming reached a peak during 1913 with 757 000 birds (De Jager, 1985, personal communication). Currently (1985) there are approximately 120 000 domesticated ostriches in South Africa. The commercial production of ostrich meat started in 1958 and the first abattoir was built in 1964. At the present moment 85 000 birds are slaughtered anually. Although the income from the carcass of a 14 month old ostrich comprise only 15% of the total income of a 14 month old bird (With the hide comprising 65% and, the first harvest feathers 20%), the subjects of carcass characteristics and meat quality has long been neglected. Very little is known about the anatomy of the skeleton and muscles of the ostrich, the post mortem reactions of these muscles and the effects of different slaughtering, chilling and deboning practices. The necessity of the above mentioned have proved very usefull in the beef, sheep, pork and poultry industries. With this present study we described the essential skeletal and muscular anatomy and included well known practices to the meat industry, such as electrical stimulation of the carcasses, post mortem pH monotoring of certain muscles, different deboning practices and vacuum packed ageing of selected cuts of ostrich meat. The obtained results are in agreement with general meat science, with expected inter-species variation, e.g. where the final pH-values of pork is relatively low at approximately 5,5; that of beef is higher at 5,7 and ostrich meat even higher at 6,0. Certain other fields of essential research are pointed out in this study, such as the growth and development of the ostrich, as well as certain basic biochemical research on the meat of the ostrich.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The ostrich industry in South Africa is currently threatened by respiratory disease in feedlot ostriches which causes a dramatic loss in production. Ms01, Ms02 and Ms03 were identified as the three ostrichspecific mycoplasmas to be associated with this respiratory disease in ostriches of South Africa. The ostrich-specific mycoplasmas have a major impact on ostrich production and for this reason there is a serious need for treatment for these infections. For this reason, the ostrich industry has undertaken an investigation into the development of vaccines against mycoplasma infections. In this study, an approach to DNA vaccine development will be investigated and applied, specifically for the ostrich mycoplasma Ms02. Firstly, the whole genome of Ms02 was sequenced using GS FLX sequencing technology. The contiguous sequences obtained from the whole-genome sequencing were analysed bioinformatically which included the annotation of the contiguous sequences and the subsequent search for a vaccine candidate gene for the development of a DNA vaccine. The P100 gene of Ms02, which showed a high degree of homology with the P100 gene of the human pathogen M. hominis, was chosen as a vaccine candidate gene for the development of a DNA vaccine. The P100 gene was successfully cloned and subsequently modified by means of site-directed mutagenesis to correct for alternative codon usage, where after the modified P100 gene was subcloned into the mammalian expression vector, pCI-neo for vaccination trials in the near future. / AFRIKAANSE OPSOMMING: Die volstruisbedryf van Suid-Afrika is tans bedreig deur 'n respiratoriese siekte in voerkraal volstruise wat lei tot aansienlike verliese in volstruisproduksie. Ms01, Ms02 en Ms03 is geïdentifiseer as die drie volstruis-spesifieke mikoplasmas wat 'n rol speel in hierdie respiratoriese siektes van volstruise in Suid- Afrika. Die drie volstruis-spesifieke mikoplasmas het 'n groot impak op die produksie van volstruise en om hierdie rede is daar 'n ernstige behoefte aan 'n behandeling van hierdie infeksies. Ten einde mikoplasma infeksies in volstruise te voorkom, het die Suid-Afrikaanse volstruisbedryf 'n ondersoek geloods na moontlike strategieë vir entstof ontwikkeling. In hierdie studie, is 'n benadering van DNA entstof ontwikkeling ondersoek en toegepas, spesifiek teen die volstruis mikoplasma Ms02. Eerstens, is die volledige Ms02 genoomvolgorde bepaal deur gebruik te maak van GS FLX volgordebepalingstegnologie. Die gedeeltelike volgordes verkry vanaf die heelgenoom volgordebepaling is bioinformaties geanaliseer wat die annotering van die gedeeltelike volgordes asook die soektog vir 'n kandidaat entstof geen vir die ontwikkeling van 'n DNA entstof ingesluit het. Die P100 geen van Ms02, wat hoë homologie met die P100 geen van die menslike patogeen M. hominis getoon het, is gekies as die kandidaat entstof geen. Die P100 geen is suksesvol gekloneer en gemodifiseer deur middel van setelgerigte mutagenese om die P100 geen geskik te maak vir die invoeging in die soogdier ekspressie vektor, pCI-neo vir toekomstige entstofproewe.

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

A criação industrial de avestruzes no Brasil tem crescido nos últimos anos, mas avestruzes podem ser reservatórios de agentes com potencial zoonótico, como é o caso do vírus da influenza, rotavírus, vírus da encefalomielite eqüina do leste (EEEV) e do oeste (WEEV), salmonela, leptospira e micoplasma. O objetivo deste trabalho foi detectar rotavírus nas fezes de filhotes, anticorpos contra rotavírus, EEEV e WEEV, influenza A , leptospira, salmonela e micoplasma a partir do soro de reprodutores e efetuar o isolamento de Salmonella sp. a partir de de suabe cloacal. Para a detecção de rotavírus nas fezes utilizaram-se as técnicas de PAGE, isolamento viral e genotipagem pela RT-PCR. As técnicas de contraimunoeletroosmoforese, soroneutralização em cultura de células, inibição da hemaglutinação e aglutinação em placa foram utilizadas nos levantamentos sorológicos e a cultura bacteriológica foi utilizada para detecção de Salmonella spp. em suabes de cloaca. Rotavírus do grupo A com genotipos G[6], G[10], P[1] e P[7] foram detectados nas fezes de avestruzes e anticorpos anti-rotavírus em 10 amostras (10/182) de soros colhidos de avestruzes reprodutores. Foram detectados anticorpos para vários sorovares de Leptospira spp. (19/128), Salmonella pullorum (17/182), Mycoplasma gallisepticum (17/182) e Mycoplasma synoviae (47/182). Não foram detectados anticorpos anti-EEEV e WEEV e anti-vírus da Influenza A H3, bem como amostras de Salmonella spp. em suabes de cloaca. Estes resultados permitem sugerir o papel do avestruz na cadeia epidemiológica das rotaviroses e da leptospirose e dão bases para o aprimoramento sanitário do plantel brasileiro desta ave. / The industrial ostrich breeding in Brazil has grown in the last few years, but ostrich may be reservoirs of potentially zoonotic agents such as influenzavirus, rotavirus, eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV), Salmonella, Leptospira and Mycoplasma. The aim of the present study was to detect rotavirus in fecal samples of young ostriches, antibodies against rotavirus, EEEV and WEEV, influenza A , Leptospira, Sallmonela and Mycoplasma in sera from breeders and carry out Salmonella isolation in cloacal swabs. For the rotavirus detection, PAGE, viral isolation and genotyping by RT-PCR were used. Counterimmunoelectroosmophoresis, virus neutralisation assay in cell culture, hemagglutination inhibition and plate agglutination were used in the serological surveys and bacteriological culture was used to detetc Salmonella spp. in the cloacal swabs. Group A rotavirus with genotypes G[6], G[10], P[1] and P[7] was detected in the fecal samples and anti-rotavirus antibodies were detected in ten samples (10/182) from breeder ostriches. Antibodies to different serovars of Leptospira spp. (19/128), Salmonella pullorum (17/182), Mycoplasma gallisepticum (17/182) and Mycoplasma synoviae (47/182) were detected. Antibodies against EEEV, WEEV and influenzavirus A H3 were not detected, as well as Salmonella spp. in swab samples. These results allow one to suggest a role to ostriches in the epidemiological chain of rotavirus diseases and leptospirosis and give basis to the improvement of the sanitary condition of the Brazilian flocks.

O objetivo deste trabalho foi estudar os níveis séricos dos hormônios esteróides (estradiol e progesterona) e analisar as possíveis relações destes com o desenvolvimento do comportamento reprodutivo em fêmeas jovens de avestruzes (Struthio camelus) durante a maturação sexual. No primeiro experimento, duas fêmeas jovens foram avaliadas, três vezes por semana, através de colheitas de sangue para dosagem de estradiol e progesterona e de observações para avaliação do comportamento reprodutivo. No segundo experimento, 20 fêmeas jovens foram selecionadas de acordo com a intensidade de comportamento reprodutivo e tiveram seu comportamento reprodutivo monitorado semanalmente. Foram realizadas colheitas de sangue semanais nos mesmos dias das observações comportamentais. No primeiro experimento, através da comparação das médias dos valores de estradiol nas fases de presença (CCOMP) ou ausência de comportamento reprodutivo (SCOMP), verificou-se uma igualdade entre as médias nas duas fêmeas estudadas e não foi verificada correlação entre os níveis séricos de estradiol e o comportamento reprodutivo. No segundo experimento, a comparação das médias de estradiol entre as amostras dos animais SCOMP e CCOMP apresentou diferença significativa. A comparação das médias entre as 4 categorias comportamentais estabelecidas - sem comportamento (SCOMP), com baixo comportamento (COMPBX), com alto comportamento sem aceitação de cópula (COMPALTSACC), com alto comportamento com aceitação de cópula (COMPALTCACC) - demonstraram haver diferença significativa entre os valores de estradiol encontrados, também sendo verificada correlação entre as 4 categorias e os níveis médios de estradiol (r = 0,64). Para a progesterona, no primeiro experimento, os valores médios foram significativamente maiores nas amostras SCOMP do que nas CCOMP, sendo que a progesterona apresentou uma correlação negativa com a intensidade do comportamento reprodutivo nas duas fêmeas (r = -0,54 e r = -0,33). No segundo experimento, os valores médios de progesterona foram significativamente maiores nas amostras CCOMP do que nas amostras SCOMP. As médias de progesterona das 4 categorias comportamentais apresentaram diferenças significativas e houve correlação positiva (r = 0,43) dos níveis de progesterona com as 4 categorias comportamentais. Os resultados sugerem que exista uma relação entre os níveis de estradiol e progesterona e a modulação da intensidade de comportamentos reprodutivos, mas não necessariamente entre estes níveis hormonais e a iniciação destes comportamentos e que os perfis séricos de estradiol e progesterona em fêmeas de avestruz na fase de maturação sexual apresentam semelhanças com outras espécies de aves. / The purpose of this work was to study the serum levels of estradiol and progesterone and analyze their relationship with the development of the reproductive behavior in young female ostriches (Struthio camelus) during sexual maturity. The first experiment studied two young females, through blood samples, three times a week, for measurement of estradiol and progesterone and was performed behavioral observations at the same time. In the second experiment, twenty young female ostriches had their reproductive behavior monitored weekly and blood samples were collected for hormone assays in the same days of the observations. The results of the first experiment showed no differences between the mean of estradiol levels in the two phases, with and without reproductive behavior. In the same way there were no correlations between estradiol levels and frequency of behaviors. In the second experiment, there were significant differences between the mean of estradiol levels with and without reproductive behavior. When it was considered four categories of behavior (without behavior, low behavior, high behavior and no copulation posture, and high behavior and copulation posture display), the mean estradiol levels showed significant differences. There was positive correlation between the categories and the estradiol levels (r = 0,64). Regarding progesterone, the first experiment, demonstrated significantly higher hormonal levels in the absence than in the presence of reproductive behavior and a negative correlation between the occurrence of behaviors and the hormonal levels for both females (r = -0,54 and r = -0,33). In the second experiment, the means of progesterone levels were significantly higher in the phase with behavioral displays than with none. For the four behavioral categories, the means of progesterone levels differed significantly and showed a positive correlation (r = 0,43) with the reproductive behavior. The results suggest that there is a relationship between estradiol and progesterone levels and the occurence of the reproductive behavior, but not necessarily between these hormone levels and the initiation of the process. The hormonal levels of estradiol and progesterone found in ostriches were similar to some domestic avian species.