The natural reservoir of influenza A virus (IAV) is wild waterfowl, and all human IAVs have their genetic origins from avian viruses. Neuraminidase inhibitors (NAIs) are currently the best drugs for treatment of human influenza; therefore, the orally available NAI oseltamivir (Tamiflu®) has been stockpiled worldwide as part of pandemic preparedness planning. Re-sistance to NAIs is related to worse clinical outcomes and if a new pandemic influenza virus would be oseltamivir-resistant its public health impact would be substantially worsened. The active metabolite oseltamivir carboxylate (OC) is not removed by sewage treatment and ends up in river water, where OC-concentrations up to 0.86µg/L have been detected. We hypothesize that occasional OC exposure of wild waterfowl carrying IAVs may result in circulation of resistant variants that may potentially evolve to become human-pathogenic. We tested the hypothesis in an in vivo Mallard (Anas platyrhynchos) model in which birds were infected with avian IAVs and exposed to OC. Excreted viruses were analyzed regarding genotypic and phenotypic resistance by neuraminidase (NA) sequencing and a functional NA inhibition assay. Two viruses with NAs of the phylogenetic N2-group, H6N2 and H7N9, acquired the NA substitutions R292K and I222T when host ducks were exposed to 12µg/L and 2.5µg/L of OC, respectively. Drug susceptibilities were at previously described levels for the substitutions. To test persistence of resistance, an OC resistant avian H1N1/H274Y virus (with a group N1 NA-protein) from a previous study, and three resistant H6N2/R292K variants were allowed to replicate in Mallards without drug pressure. Resistance was entirely maintained in the H1N1/H274Y virus, but the H6N2/R292K variants were outcompeted by wild type virus, indicating retained fitness of the resistant H1N1 but not the H6N2 variants. We conclude that OC in the environment may generate resistant IAVs in wild birds. Resistant avian IAVs may become a problem to humans, should the resistance trait become part of a new human pathogenic virus. It implies a need for prudent use of available NAIs, optimized sewage treatment and resistance surveillance of avian IAVs of wild birds.

Domestication in chickens has made feed-restriction a necessity if broiler breeder hens should reach sexual maturity and be fertile. This is claimed to cause chronic hunger. To measure hunger the gene expression of the appetite regulators agouti-related peptide (AgRP), pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and adenosine monophosphate-activated protein kinase (AMPK) of the melanocortin system was quantified with qPCR. This was done in feed-restricted Red Junglefowl and compared with the gene expression of two strains of feed-restricted broilers, Ross 308 and Rowan Ranger, to detect possible effects on domestication on appetite regulation. POMC-expression was upregulated 2-fold in the feed-restricted Red Junglefowl. POMC-expression was downregulated by half in the feed-restricted Ross 308. AgRP/NPY-expression was upregulated 4-fold in feed-restricted Rowan Rangers. A comparison between the control groups (ad libitum fed) of the breeds showed that the NPY-expression was lower in Ross 308 and Rowan Ranger compared with the ancestor. Results show no difference in body weight of ad libitum fed and feed-restricted Red Junglefowl. Conclusions were that the feed-restricted Red Junglefowl was not properly restricted in food supply since no difference in body weight between the treatment groups was detected. The upregulation of POMC in the feed-restricted Red Junglefowl could be stress-linked influenced by the feeding type (scattering of food in litter). No conclusions of the impact of domestication on chicken’s appetite could be drawn. Domestication has probably had its impact by altering other signaling pathways of the melanocortin system than in the arcuate nucleus.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The African penguin, which occurs along the south-eastern and south-western shores of South-Africa and Namibia, has experienced a severe reduction in population numbers due to guano and egg collection in the first half of the 19th century, and oil pollution in the second half of the 19th century as a result of oil tankers rounding the Cape of Good Hope. The population would have been reduced by a further 19% had it not been for the rehabilitation of penguins at the South African National Council for the Conservation of Coastal Birds (SANCCOB) facility. Although this has been very successful, mortalities as a result of avian malaria infection have considerably reduced the efficiency of rehabilitation. In an effort to assess the role of immunity against malaria in combating the disease, an enzyme-linked immunosorbent assay (ELISA) for the detection of antibody levels to avian malaria was developed. The ELISA was used to detect antibody levels to avian malaria of penguins on entry and during rehabilitation from October 2001 to January 2003. The aim of this study was to continue the determination of antibody levels to avian malaria of penguins entering the SANCCOB facility, in order to allow an evaluation of the antibody levels to avian malaria for two full calendar years. This investigation was combined with a polymerase chain reaction (PCR)-based method, capable of detecting any Plasmodium species in penguin serum. These two methods were also used to investigate avian malaria in several breeding colonies in order to assess the role avian malaria may play in the survival of the African penguin in the wild. Results indicated that the ability of penguins to produce anti-Plasmodium antibodies was not influenced by oiling and that infection with malaria was not due to recrudescence but rather due to infection via mosquitoes. This indicated a possible role of the SANCCOB facility in exposing the penguins to avian malaria. However a large number of penguins arrived at the facility previously infected with malaria, indicating that malaria was present in the breeding colonies. Investigations in the breeding colonies revealed extremely high avian malaria prevalence even though no sick birds or mortalities were observed. This raised the question whether different types of malaria are responsible for infection in the SANCCOB facility and breeding colonies. / AFRIKAANSE OPSOMMING: Die Afrika Pikkewyn kom langs die suid-oostelike en suid-westelike kus van Suid Afrika en Namibië voor. In die afgelope eeu het hierdie spesie ‘n geweldige afname in populasie getalle ondervind. Dit was hoofsaaklik die gevolg van die versameling van guano en pikkewyneiers in die eerste helfte van die 19de eeu en oliebesoedeling in die tweede helfde van die 19de eeu. Die “South African Foundation for Conservation of Coastal Birds” (SANCCOB) is ‘n seevoëlreddings- en rehabilitasiesentrum vir siek, beseerde en ge-oliede pikkewyne. Dit word geskat dat die Afrika Pikkewyn populasie met ‘n verdere 19% sou afgeneem het as dit nie vir die rehabilitasie by die SANCCOB sentrum was nie. Hierdie sentrum het egter aansienlike vrektes in die somer as gevolg van voëlmalaria, wat sodoende die effektiwiteit van die rehabilitasie verlaag. In ‘n poging om die rol van immuniteit teen malaria te bepaal is ‘n “enzyme-linked immunosorbent assay” (ELISA) ontwikkel vir die bepaling van antiliggaam vlakke teen malaria. Hierdie ELISA is gebruik vir die bepaling van die anti-Plasmodium antiliggaam vlakke van die pikkewyne by aankoms en ten tye van rehabilitasie by SANCCOB vanaf Oktober 2001 to Januarie 2003. Die doel van hierdie studie was eerstens om hierdie ELISA bepalings voort te sit om sodoende antiliggaam vlakke teen malaria oor twee kalender jare te kan evalueer. Hierdie ondersoek was gekombineer met ‘n polimerase ketting reaksie (PCR) metode, wat enige Plasmodium spesie in pikkewynserum sou kon opspoor. Hierdie twee metodes is ook gebruik vir ondersoeke in sommige broeikolonies, met die doel om te bepaal watter rol voëlmalaria in die oorlewing van die Afrika pikkewyn in die natuur speel. Resultate het getoon dat olie nie die vermoë van die pikkewyn beïnvloed om anti- Plasmodium antiliggame te vervaardig nie en dat malaria infeksie hoofsaaklik deur muskiete veroosaak word en nie deur heruitbraak van ‘n bestaande infeksie nie. Dit dui egter daarop dat pikkewyne blootgestel word aan voëlmalaria by die SANCCOB sentrum. Daar is ook gevind dat ‘n groot aantal pikkewyne met malaria infeksies by die sentrum opgedaag het wat dui op die voorkoms van malaria in die broeikolonies. Ondersoeke in die broeikolonies het ‘n besonder hoë voorkoms van malaria onthul. Geen vrektes of siek pikkewyne is in die broeikolonies waargeneem nie, wat moontlik kan beteken dat pikkewyne by SANCCOB met ‘n ander tipe malaria geïnfekteer word as in die broeikolonies.

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

Effects of dietary supplementation with low levels of organic sources of trace minerals in place of normal levels of their inorganic salts and phytase on growth performance and mineral metabolism were evaluated in two studies using pullets of white and brown shell laying strains. The organic sources were proteinates of copper, iron, manganese and zinc and selenium yeast. A corn-soybean meal diet was fed alone, plus inorganic minerals or plus organic minerals, and with or without phytase in a 3 x 2 factorial arrangement. Twelve groups of 16 pullets, 2 weeks old, were used per treatment. Compared with inorganic minerals, feeding no mineral supplement or organic minerals significantly (P<0.05) decreased manure Cu, Fe and Zn for white pullets and Cu, Fe, Mn and Zn for brown pullets. Dietary phytase significantly reduced manure Fe, P and Ca for white pullets and Fe, Mn, Zn, P and Ca for brown pullets. Adding phytase to diets containing inorganic minerals reduced manure Zn concentration for white pullets and manure Fe, Mn, Zn, P and Ca concentrations for brown pullets. These studies indicate manure levels of trace minerals can be decreased by using low levels of organic mineral supplements and phytase in pullet diets.

published_or_final_version / Environmental Management / Master / Master of Science in Environmental Management

published_or_final_version / Community Medicine / Master / Master of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Cooperative oxygen binding by vertebrate tetrameric hemoglobins (Hbs) has been extensively studied and is relatively well understood. Nonetheless, Hill coefficients greater than four have been reported for adult avian, amphibian, and reptilian red blood cells. Such reports also exist for embryonic red cells from various animals. These results are controversial and not yet convincingly established. Oxygen binding studies on avian Hb D, which is known to undergo deoxygenation-dependent self-association, were carried out to answer this question. The goal was to determine unequivocally whether Hill coefficients greater than four occur. Such high Hill coefficients were observed but only at very high Hb D concentrations. Moreover, the early model of avian deoxy Hb D self-association was found to be incomplete. The model has now been expanded to describe better the observed sedimentation data at high Hb concentrations. The possibility that embryonic deoxy Hbs self-associate was also assessed by sedimentation studies of deoxygenated Hb solutions from a marsupial, the tammar wallaby. The results obtained show unambiguously that these embryonic Hbs self-associate upon deoxygenation. Recent phylogenetic analyses suggest that the avian [alpha superscript D]-globin originated from embryonic [alpha]-globins. This finding suggests that the propensity to self-associate upon deoxygenation is an intrinsic property of tetrameric Hbs with embryonic [alpha]-globins. Furthermore the residues mediating the inter-tetramer interactions in adult avian deoxy Hb D and embryonic deoxy Hbs are likely to be the same. Recombinant globins were expressed in bacteria and protocols for the assembly of avian recombinant tetrameric Hb D developed. Initial measurements by sedimentation were carried out to verify the role of a conserved glutamate residue previously speculated to be involved in inter-tetramer interactions. The present studies provide a framework for future investigations of deoxygenation-dependent Hb self-association. In particular the need to carry out oxygen equilibrium measurements at high Hb concentrations as well as sedimentation studies of the deoxygenated Hb solutions is stressed. / text