Rozprawy doktorskie na temat „Microorganisms disease”

The ability to mount an efficient immune response should be an important life-history trait as parasitism can impact upon an individual's fecundity and survival prospects, and hence its fitness. However, immune function is likely to be costly as resources must be divided between many important traits. Whilst many studies have examined host resistance to particular parasite types, fewer have considered general immune responses. Studies that have considered general immune responses tend to do so in vertebrate models. However, the complexity of the vertebrate immune system makes the examination of evolutionary aspects of immune function difficult. Using larvae of the genus Spodoptera (Lepidoptera: Noctuidae) as a model system, this study examines' genetic and phenotypic aspects of innate immunity. The aims were to assess the levels of additive genetic variation maintained in immune traits, to consider possible costs that could maintain this variation, and to assess the role of phenotypic plasticity in ameliorating those costs. A key finding of this study was that high levels of additive genetic variation were maintained in all of the measured Immune traits. Analysis of the genetic correlations between traits revealed potential trade-offs within the immune system and between immune components and body condition. In addition, it was shown that larvae living at high densities invest more in immune function than those living in solitary conditions, suggesting that larvae can minimise the costs of immune function by employing them only when the risk of pathogenesis is high.

Prion diseases or transmissible spongiform encephalopathies (TSE) are characterised by the accumulation of a misfolded conformer (PrPSc) of a host encoded protein (PrPC). The misfolding event that leads to the formation PrPSc can be replicated in the in vitro amplification technique, protein misfolding cyclic amplification (PMCA). This thesis focuses on the application PMCA to study multiple aspects of prion misfolding in relation to ruminant prion diseases, specifically developing techniques to detect and characterise PrPSc in scrapie and BSE infections. Utilising recombinant hamster PrP (rPrP) as substrate in PMCA, multiple genotypes of scrapie were successfully amplified in an attempt to describe a quantifiable technique applicable to a wide range of scrapie isolates. Observations of non-specific protease resistant rPrP formation was investigated with modifications to the PMCA methodology, which ultimately proved unsuccessful in reducing non-specific protease resistant rPrP. Using brain PrPC as substrate, the quantitative PMCA technique was piloted with BSE to correlate in vitro replication efficiency with infectious titre in mouse bioassay, but no correlation was identified. Atypical forms of BSE occur primarily in older cattle, are asymptomatic and thought to be spontaneous diseases. None the less, infection models in rodents and primates have identified the zoonotic potential of H-type and L-type BSE. Therefore PMCA methods were developed which were able to successfully amplify both atypical forms of BSE. In particular, sensitive detection and discrimination from classical BSE was demonstrated for H-type BSE, which has not previously been amplified in PMCA. H-type BSE could be detected in 1x10¬-12 g brain material and was discriminated from classical BSE by increased protease sensitivity, relatively high molecular weight and antibody reactivity. Evidence exists for co-infection of TSE strains, yet scrapie and BSE co-infection in an ovine host remains unaddressed. To study the disease progression and tissue dissemination of co-infections a PMCA assay capable of specifically amplifying BSE PrPSc in the presence of excess scrapie was applied to artificially mixed brain homogenates containing BSE and scrapie, and compared to current statutory strain typing methods. The PMCA was found to have sensitivity and specificity of 100% in mixes containing 0.1% BSE and 99.9% scrapie brain material, which was more effective than conventional strain typing methods. The assay was then applied to the brain, spleen and lymph of scrapie and BSE experimental co-infections in two genotypes of sheep, and to animals which belonged to a flock with endemic natural scrapie and that also received experimental BSE infections. The PMCA data demonstrated that sheep with PRNP genotype ARQ/ARQ (at amino acid positions 134, 154 and 171) were resistant to BSE in a co-infection scenario. In sheep with PRNP genotype of VRQ/ARQ, mixed infections could occur, and animals with scrapie PrPSc only in the brain could harbour BSE PrPSc in peripheral tissues. Co-infection was also possible in sheep with natural scrapie infections. The assay was compared to conventional testing methods of western blotting, PrPd profiling and immunohistochemistry and displayed superior sensitivity in BSE detection. PMCA amplification of bovine BSE isolates in ovine substrates identified several instances in which the molecular characteristics of the PrPSc was scrapie-like in terms of molecular weight, antibody reactivity and glycoform profile, and in some cases PrPSc characteristic of BSE could no longer be recovered. This occurred in a genotype specific manner, ‘molecular switching’ was only apparent in ovine substrate VRQ/VRQ in accordance with previous findings. These results raise the possibility of such an event occurring in in vivo ovine BSE infections and the zoonotic potential of these scrapie like conformers are yet to be fully addressed.

Diarrhoeal disease is a major cause of mortality in children in developing countries. It also remains a serious problem among all age groups throughout the world. Whereas studies to determine the epidemiological and aetiological factors of diarrhoeal disease have been reported for other parts of South Africa and the world, as yet no information is available for the Eastern Cape. Therefore this study was undertaken to determine the factors for this area. Enteropathogens were compared for the different ages in the various population groups and, where possible, seasonal and geographical differences were emphasised. A total of 7 278 faecal samples were examined by six laboratories in the Eastern Cape during the period November 1988 to October 1990. Data was recorded noting the age, sex and population group of the patients. The towns selected were Port Elizabeth, Uitenhage, Cradock, Grahamstown and their surrounding areas. The isolation rates for the pathogens studied in the various population groups were compared to those reported in similar studies in other countries. The seasonal incidences of the various selected pathogens were compared with those reported from elsewhere in South Africa. It was thought that the higher temperature of summer may influence the finding in the White population group, while rainfall would play a greater role for the Coloured and Black populations. The geographical distribution of the pathogens emphasised the difference in living conditions between the different population groups. For example a generally higher infestation rate of Helminths occurred in rural areas and in the groups living under poorer conditions. The low isolation rates for certain bacteria and the large percentage of samples from which no pathogens were isolated indicate the need for further research. However, the finding should be valuable for determining Public Health priorities and in the management of outbreaks of diarrhoeal disease.

Thesis (MScAgric)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Petri disease and esca are devastating grapevine trunk diseases and compromise the sustainability of viticulture world-wide. Despite being extensively studied, knowledge of inoculum sources and mechanisms of spread of the causal pathogens is limited. Arthropods have been suspected to play a role in the spread of Petri disease and esca pathogens. However, little information is known about the extent to which arthropods are associated with these pathogens. This study aimed to determine whether arthropods occurring within or on declining grapevines, are associated with trunk disease pathogens and to identify arthropods associated with pruning wounds. The potential of selected arthropods to act as vectors of trunk disease pathogens was also investigated. Two vineyards exhibiting grapevine trunk disease infections were sampled weekly for two years for collection of arthropods. Arthropods were collected using pruning wound traps, visual searches as well as trunk and cordon traps. Fungal spores from surfaces of arthropods were collected in water. Samples were subjected to nested PCR using primers Pm1/Pm2 and Pch1/Pch2 to verify the presence of Phaeoacremonium spp. and Phaeomoniella chlamydospora, respectively. Water samples were also cultured and grapevine trunk disease pathogens obtained were identified by sequencing the internal transcribed spacers 1 and 2 and the 5.8S rRNA gene or the partial beta-tubulin gene. A total of 10 875 arthropod individuals, belonging to more than 31 families, were collected from declining grapevines. The most abundant arthropods included millipedes, ants, spiders and beetles. Portuguese millipedes and cocktail ants were associated with fresh grapevine pruning wounds. Thirty-three percent of the 5677 water samples analysed, contained propagules of pathogens associated with Petri disease and esca. Of these, 37 % were recovered from millipedes, 22 % from cocktail ants, 15 % from spiders and 10 % from beetles. All the major groups of grapevine trunk diseases were detected on the arthropods. Phaeoacremonium species were detected in 1242 samples while Phaeomoniella chlamydospora was identified from 855 samples. Other fungi isolated included members of the Botryosphaeriaceae, Diatrypaceae and Diaporthales. The potential of grapevine sap as a food source for Portuguese millipedes and cocktail ants was investigated, in vitro. Millipede individuals were offered a choice between water and grapevine sap while ants in nests were presented with grapevine sap, tuna and water and monitored for ingestion of sap. Both taxa preferred grapevine sap over the other food items, indicating close association with pruning wounds. Subsequently, the ability of both taxa to transmit a DsRed-transformed Phaeomoniella chlamydospora isolate to fresh pruning wounds of canes in polystyrene strips, floating in water, and potted vines was tested. Arthropods were exposed to the fungus for 24 hours and transferred to the base of the plants and canes and were removed after three days. Isolations after a month revealed that millipedes and ants were capable of transmitting the fungus onto wounds and cause infection. Millipede faecal pellets were also evaluated as potential sources of inoculum. Millipedes were fed on Phaeomoniella chlamydospora for 24 hours, surface sterilised and allowed to defaecate in sterile Petri dishes overnight. Faecal material was collected, macerated in water and plated onto potato dextrose agar. Propagules of Phaeomoniella chlamydospora survived passage through the gut of millipedes and were passed out in a viable state to form colonies of Phaeomoniella chlamydospora. This study concludes that a wide variety of arthropods can be a source of inoculum of trunk diseases in vineyards. The results of the dissemination trial provides evidence that millipedes and ants are able to disseminate and infect vines with Phaeomoniella chlamydospora. It is therefore, highly likely that other grapevine trunk disease pathogens are transmitted in the same manner. This knowledge highlights the need for control of certain arthropods to be taken into consideration when managing grapevine trunk disease pathogens.
AFRIKAANSE OPSOMMING: Petri siekte en esca is verwoestende wingerd stamsiektes en verhinder die volhoubaarheid van wingerdproduksie wêreldwyd. Hierdie siektes is al intensief bestudeer, maar kennis rakende die inokulum bronne en meganismes van verspreiding van die veroorsakende patogene is beperk. Arthropoda is al vermoed om ‘n rol te speel in die verspreiding van Petri siekte en esca patogene, maar weinig informasie is bekend oor die mate waartoe arthropoda geassosieer is met die patogene. Hierdie studie het ten doel gestel om die arthropoda wat op of in wingerdstokke wat terugsterf voorkom te identifiseer en te bepaal watter van die arthropoda geassosieer is met stamsiekte patogene. Daar is ook ten doel gestel om die arthropoda wat geassosieer is met vars snoeiwonde te identifiseer en ook die moontlike vektor status van die stamsiekte patogene deur arthropoda. Arthropoda is weekliks vir twee jaar gekollekteer vanaf twee wingerde met stamsiekte infeksies. Snoeiwond lokvalle, visuele soektogte en stam- en kordon lokvalle was gebruik om arthropoda te vang. Swamspore van die oppervlak van die arthropoda is afgewas met water. Van hierdie water monsters is gebruik om dubbelvoudige polimerase ketting reaksies (PKR) te doen met die inleiers Pm1/Pm2 en Pch1/Pch2 om vir die teenwoordigheid van Phaeoacremonium spp. en Phaeomoniella chlamydospora onderskeidelik te toets. Die oorblywende water monster is gekweek op medium om die swamme teenwoordig te bepaal. Die wingerd stamsiekte patogene is verder geidentifiseer deur die DNS volgordes te bepaal van die interne getranskribeerde spasies 1 en 2 en die 5.8S rRNS geen of ‘n gedeelte van die beta-tubulien geen. In totaal is 10 875 arthropoda, wat behoort tot 31 families, gekollekteer vanaf wingerde wat terugsterf. Die mees algemene arthropoda was duisendpote, miere, spinnekoppe en kewers. Die Portugese duisendpote en die wipstert mier is geassosieer met vars wingerd snoeiwonde. Van die 5677 water monsters wat geanaliseer is, het 33% propagules van die Petri siekte of esca patogene gehad. Van hierdie was 37 % afkomstig vanaf duisendpote, 22 % van wipstert miere, 15 % van spinnekoppe en 10 % van kewers. Al die hoofgroepe van wingerd stampatogene is opgespoor op die arthropoda. Phaeoacremonium species is opgespoor in 1242 monsters en Phaeomoniella chlamydospora is gevind in 855 monsters. Ander swamme wat ook geisoleer is sluit lede van die Botryosphaeriaceae, Diatrypaceae en Diaporthales in. Die potensiaal van wingerdsap as ‘n bron van voedsel vir Portugese duisendpote en wipstert miere is in vitro ondersoek. Duisendpoot invidue is ‘n keuse gegee tussen water en wingerd sap terwyl mierneste ‘n keuse gehad het tussen water, wingerd sap en tuna. Die duisendpote en miere is gemonitor vir die inname van wingerdsap in die teenwoordigheid van die ander bronne. Beide die duisendpote en miere het wingerdsap verkies wat aandui dat hulle ‘n noue assosiasie met wingerd snoeiwonde het. Vervolgens is beide taksons getoets vir hul vermoë om ‘n DsRooi-getransformeerde Phaeomoniella chlamydospora isolaat te vektor na vars snoeiwonde op lote gemonteer op polistireen stroke wat in water dryf en op wingerd plante in potte. Die duisendpote en miere is blootgestel aan die swam vir 24 uur en oorgedra na die basis van die plante en lote en is weer verwyder na drie dae. Na ‘n maand is isolasies gedoen wat gewys het dat die duisendpote en miere die swam suksesvol kon oordra na die snoeiwonde en infeksie veroorsaak. Duisendpoot uitwerpsels is geëvalueer vir die potensiaal as inokulum bron. Duisendpote het gevoed op Phaeomoniella chlamydospora vir 24 uur, daarna oppervlakkig gesteriliseer en toegelaat om oornag uitwerpsels te maak in steriele Petri bakkies. Uitwerpsels was gekollekteer, fyngemaak in water en op aartappel dekstrose agar uitgeplaat. Propagules van Phaeomoniella chlamydospora het die verteringskanaal van die duisendpote oorleef en het tipiese kolonies op die agar gevorm. Hierdie studie het vasgestel dat ‘n verskeidenheid van arthropoda ‘n bron van inokulum van stamsiektes in wingerd kan wees. Die resultate van die vektor proewe het gewys dat duisendpote en miere die vermoë het om Phaeomoniella chlamydospora te versprei na snoeiwonde wat die swam dan suksesvol geinfekteer het. Dit is daarom hoogs waarskynlik dat van die ander wingerd stamsiekte patogene ook versprei kan word op dieselfde manier. Hierdie kennis demonstreer dat die beheer van spesifieke arthropoda in ag geneem moet word in die bestuur van wingerd stamsiektes.
Winetech, Agricultural Research Council of South Africa and NRF for financial support

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Com o objetivo da redução da aplicação de fungicidas na produção de alho, foram estudados os efeitos dos Microrganismos Eficazes (E.M.-4 e E.M.-5) sobre o controle da mancha púrpura, a produção da cultura e as propriedades físicas, químicas e biológicas do solo. Os experimentos foram conduzidos, em condições de campo, por dois e três anos, na Fazenda de Ensino, Pesquisa e Produção da Unesp em São Manuel, SP. O E.M. foi aplicado nos bulbilhos de alho antes do plantio, adicionado ao material orgânico incorporado ao solo e pulverizado nas plantas após incubação ou não com melaço. A utilização do E.M. + melaço (não incubado) não proporcionou controle da doença nos experimentos. No entanto, com a incubação do E.M. + melaço houve redução na severidade da mancha púrpura em um experimento e incremento na emergência e número de folhas verdes por planta em ambos os experimentos. A altura das plantas, superbrotamento, produção, bulbos de maior valor comercial (classes 5+6+7) e as propriedades físicas (densidade do solo, condutividade hidráulica, estabilidade dos agregados, resistência à penetração e infiltração de tinta), químicas e biológicas do solo (conteúdo de polissacarídeos, carbono da biomassa microbiana do solo e atividade da desidrogenase) não foram alteradas pela utilização do E.M. A adição de material orgânico ao solo promoveu maior agregação do solo (estabilidade dos agregados), independentemente dos tratamentos empregados. No entanto, a densidade do solo, a condutividade hidráulica e a resistência à penetração não sofreram alterações com a adição do material orgânico.
With the purpose to reduce fungicides application for garlic production, the effects of Effective Microorganisms (E.M.-4 and E.M.-5) were studied on the control of garlic purple blotch, garlic yield and physical, chemical and biological properties of the utilized planting soil. Experiments were carried out under field conditions, for two and three years, an experimental farm, belonging to São Paulo State University, located in São Manuel, São Paulo, Brazil. E.M. was applied on garlic cloves before planting, additioned to the incorporated organic matter and sprayed on garlic aerial part after incubation or not with molasse. When E.M. plus molasse (not incubated) was utilized the control of purple blotch was not observed in two experiments. However, with the incubation of E.M. with molasse, the results showed a reduction of purple blotch severity in one experiment as well as an increment of seedling emergency and number of green leaves per plant in two experiments. Plant height, bulbil sprouting, yield, bulbs with higher market grades (classes 5+6+7) and soil physical (bulk density, hidraulic conductivity, stable soil aggregates, resistance to soil penetration and tint infiltration) chemical and biological (polyssacharides, carbon of microbial biomass and dehydrogenase content) proprieties were not affected by E.M. utilization. Organic matter addition promoted soil agreggation in all treatments, however, soil bulk density, hidraulic conductivity and resistance to soil penetration was not changed by organic matter addition.

Introduction: Garlic-derived organosulfur compounds are associated with physiological benefits, including the reduction of cardiovascular disease (CVD) risk, possibly by reducing the risk marker trimethylamine-N-oxide (TMAO). TMAO production in humans is largely influenced by the metabolic activity of the intestinal bacteria on dietary precursors including L-carnitine. Dietary supplementation of bioactive garlic phytochemical allicin has recently been suggested to reduce the formation of TMAO precursor molecule trimethylamine (TMA) from L-carnitine through impact on the intestinal bacteria, thereby limiting the formation of TMAO by the host. Purpose: The objective of this research was to evaluate and compare the efficacy of fresh and aged garlic extracts (rich in alliin and allicin, respectively) in the reduction of circulating TMAO levels produced from L-carnitine metabolism and identify shifts in the abundances of gastrointestinal bacterial genes that may contribute to reduction in circulating TMA levels, which may, in turn, influence the levels of circulating TMAO. Methods: Five-week old female C57BL/6 mice (n = 12) were challenged with L-carnitine to assess the animal's capacity for TMAO production. Animals were gavaged daily with fresh or aged garlic extract dissolved in L-carnitine for 13 days, then challenged with L-carnitine post-treatment to evaluate changes in TMAO production. Whole blood samples were evaluated for TMAO content using UPLC-MS/MS and compared to non-extract consuming control groups. Post-mortem hepatic tissues were collected and analyzed for TMA-oxidizing flavin monooxygenase 3 (Fmo3) gene abundance and protein expression using quantitative real-time PCR (qPCR) and ELISA. Fecal samples collected prior to and following treatment were analyzed using qPCR to quantify shifts in the abundance of L-carnitine metabolizing genes cntAB and grdH. Results: Postprandial and circulating TMAO levels were not significantly affected (p < 0.05) by inclusion of garlic extract in the diet. Dietary intervention with extracts significantly increased L-carnitine-derived proatherogenic CVD risk marker γ-butyrobetaine levels ~28% higher than the increased levels observed in the positive control group supplemented with L-carnitine only. Mice administered garlic extracts had significant increases of, γ-butyrobetaine, relative to negative control mice and mice supplemented with broad-spectrum antibiotics. Mice supplemented fresh garlic extract saw a 25-fold increase in circulating γ-butyrobetaine levels after intervention; mice supplemented aged garlic extract saw a 23-fold increase in circulating γ-butyrobetaine levels after intervention. Furthermore, FMO3 protein expression levels in either extract treatment group were not significantly different (p < 0.05) from controls. Abundances of L-carnitine metabolizing genes in fecal samples of mice fed either garlic extract were not significantly higher than levels observed in positive or negative controls. Interestingly, treatment with broad-spectrum antibiotics significantly increased abundances of L-carnitine metabolizing genes cntAB and grdH when compared with controls. Abundances of hepatic Fmo3 mRNA transcript in mice supplemented garlic extracts were not significantly different from the positive control group when data were normalized to mg of liver used. Mice supplemented aged garlic extracts significantly lowered Fmo3 mRNA transcript levels relative to the negative control. Significance: This research suggests that garlic extract supplementation in conjunction with excess L-carnitine consumption may not be an appropriate dietary intervention strategy to reduce CVD risk. As it stands, garlic extract supplementation may increase CVD risk by promoting the biosynthesis of proatherogenic γ-butyrobetaine. The impact of garlic extract mediated increases in γ-butyrobetaine should be further investigated in tandem with CVD outcomes to confirm the findings presented in this study.
Doctor of Philosophy
Garlic compounds that contain sulfur are associated with many health benefits, including the reduction of heart disease risk, possibly by lowering the amount of risk marker trimethylamine-N-oxide (TMAO) in the body. TMAO is produced when the gut bacteria break down L-carnitine into trimethylamine (TMA), which is then absorbed and converted to TMAO in the liver. Garlic supplementation has recently been suggested to reduce TMAO formation, which may, in turn, reduce heart disease risk. The objective of this research was to evaluate the potential of fresh and aged garlic extracts (which have different sulfur compounds in them) to reduce TMAO levels and identify changes in the gut bacteria that may contribute to this lowering effect. Mice were fed daily with either fresh or aged garlic extract for 13 days, then given L-carnitine to evaluate changes in TMAO levels in the blood. These levels were then compared to mice that did not consume any garlic extract. Liver samples were tested for their ability to turn TMA into TMAO. Fecal samples were tested to determine if there were any changes to gut bacteria caused by the garlic extracts. TMAO levels in the mice were not significantly affected by consuming garlic extracts. Consuming garlic extracts did, however, increase another risk marker of heart disease known as γ-butyrobetaine. Feeding mice garlic extracts did not affect the ability of mice to turn TMA into TMAO, nor did it affect the gut bacteria. This research suggests that garlic extracts may not be an appropriate strategy to reduce heart disease risk. As it stands, garlic extract supplementation may increase heart disease risk by promoting the γ-butyrobetaine formation. The means that garlic extracts increase γ-butyrobetaine levels should be further investigated.

Infectious diseases have affected humanity since its apparition in Africa 300,000 years ago. Demographic changes associated to the Neolithic transition, and ensuing population movements, have facilitated the emergence and expansion of those diseases around the world. The usage of ancient DNA has allowed us to have a snapshot of ancient pathogens’ genomes. In this thesis I present the genomes of different ancient pathogens associated to a global disease, to an historical individual case and to past epidemics. In the first case, we retrieve the partial genome of a European Plasmodium falciparum strain, which hints the arrival of the parasite to Europe during antiquity. We also take a look at French revolutionary Jean-Paul Marat’s condition, in order to shed light to his mysterious condition. Finally, we analyse an ancient Salmonella enterica Paratyphi C strain, which suggests that, although infections by this particular serovar are fairly scarce in the present, in the past could have been the responsible agent of epidemics around the globe.
Les malalties infeccioses han afectat a l’ésser humà des de la seva aparició a Àfrica fa 300,000 anys. Canvis demogràfics associats a la transició neolítica, i posteriors moviments poblacionals, han afavorit l’aparició i dispersió d’aquestes malalties al voltant del mon. L’ús de ADN antic permet obtenir una finestra temporal des d’on observar com eren aquests patògens en el passat. En aquesta tesi presento els genomes d’una sèrie de patògens antics associats a malalties, casos individuals històrics i epidèmies. En el cas de la malaltia, recuperem un genoma parcial d’una soca Europea erradicada de Plasmodium falciparum, la qual dóna indicis de l’arribada del paràsit a Europa durant l’antiguitat. Fem una ullada també al cas del revolucionari francès Jean Paul Marat amb la intenció d’esbrinar l’origen de la seva condició. Finalment analitzem una soca de Salmonella enterica Paratifoide C que podria suggerir que aquest patogen, actualment escàs, era el responsable d’epidèmies al voltant del mon.

Bovine mastitis is recognised worldwide as the most important and costly disease affecting dairy cattle. The reduction of herd mastitis rates is crucially needed to improve animal welfare and profitability, and lessen the reliance on antibiotics. Single nucleotide polymorphisms (SNPs) within genes that have a critical role in the innate immune response, such as Toll-like receptors (TLRs) and the chemokine receptors CXCR1 and CXCR2, could impact on establishment and progression of intramammary infection, and therefore influence an animal’s susceptibility to disease. The genetic selection of animals with favourable TLR and CXCR1/2 mutations, with no impact on production traits, could be incorporated into dairy breeding programmes. In order to investigate any associations with clinical mastitis (CM) incidence and milk quality and quantity, this study identified and analysed SNPs alongside actual CM and production data from a Holstein-Friesian herd. This revealed 46 SNPs, 9 of which are novel, within boTLR1/4/5, boCD14, boCXCR1 and boCXCR2; selected SNPs were then tested for association with CM. This is the first report of boTLR1 SNPs and a non-coding boCXCR1 SNP that associate significantly with susceptibility to CM. Favourable linkage of reduced CM with increased milk fat and protein was observed, indicating selection for these markers would not be detrimental to milk quality. Furthermore, this study provides evidence that some of these SNPs underpin functional variation in bovine TLR1 and CXCR1, and possibly underlie an immunological mechanism for disease susceptibility. SNPs in boTLR1 and boCXCR1 were significantly associated with impaired transcript levels in milk somatic cells. In addition boTLR1 SNPs associated with impaired cytokine responses from cell populations when exposed to ligand or heat-killed mastitis-causing bacteria. The potential impact of boTLR1 variation on the immune response to Staphylococcus aureus is demonstrated, and this has implications for boTLR1-mediated immune responses to other pathogens.

It is now widely accepted that surface contamination plays an important role in the transmission of both respiratory and gastrointestinal infections in the domestic environment and community setting. The efficiency of transfer of a pathogen to the hand from a fomite is important in modeling transmission in microbial risk assessment models. The objective of this study was to use published literature to assess the role of fomites and hands in disease transmission, and to conduct fomite-to-finger transfer studies from various porous and nonporous fomites under different relative humidity condition using non-pathogenic strains of Escherichia coli, Staphylococcus aureus, MS2 coliphage, Bacillus thuringiensis spores, and poliovirus 1; to evaluate the persistence of bacteria and viruses on surfaces; to examine bacteria and virus transfer from treated surfaces; and to conduct a foodborne quantitative microbial risk assessment using Campylobacter jejuni from the data obtained in these studies. It was found that numerous factors influence the transfer efficiency of microorganisms, with moisture being the most important, with greater transfer under humid conditions. Other factors influencing transfer include drying time, contact time, pressure, friction, type of material, and porosity of the fomite. Percent transfer was greater under high relative humidity for both porous and nonporous surfaces. Most organisms on average had greater transfer under high relative humidity (40 - 65%) compared to low relative humidity (15 - 32%). Relative humidity and fomite type influenced the survival of all studied organisms; survival was greater on nonporous surfaces than those for porous surfaces. Test organisms were reduced up to 99.997% on the fomites after the surfaces were wiped with a disinfectant wipe. Microbial fomite-to-finger transfer from disinfectant wipe-treated surfaces were, lower than from non-treated surfaces. The disinfectant-wipe intervention reduced the risk of Campylobacter infection, illness, and death by 2 to 3 orders on all fomites. The disinfectant-wipe intervention reduced the annual risk of illness below the reported national average of diagnosed Campylobacteriosis cases 1.3E-04. This risk assessment demonstrates that the use of disinfectant wipes to decontaminate surface areas after chicken preparation reduces the risk of C. jejuni infections up to 99.2%.

Rhizoctonia solani (AG-3), the causal agent of Rhizoctonia disease of potato, overwinters as sclerotia on potato tubers. To develop a biocontrol strategy based on the prevention of the sclerotial germination, an isolation of microorganisms colonizing sclerotia of infected potato tubers (cultivars Norland, Atlantic and Souris), was conducted. In vitro screening was used to select effective antagonistic fungi against Rhizoctonia solani. Fifty fungal isolates were selected in order to cover all identified genera and potato variety and examined for their ability to inhibit germination of sclerotia which were incubated with the test fungus for 14 days. Twenty-four (24) fungal isolates were retained based on their ability to reduce sclerotial viability by more than 50% as compared with 100% viability of untreated sclerotia. These 24 isolates were further examined for their ability to protect Table beet seedlings against the pathogen in greenhouse soils. Based on their ability to protect Table beet seedlings from Rhizoctonia infections and to increase the number of secondary roots and root length isolates, F2, F11, F132, F158, and F258 were screened and test their efficacy to increase beet seed germination in field soils. (Abstract shortened by UMI.)

Thesis (MScAgric (Plant Pathology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: A survey was undertaken on apple and pear trees in the Western Cape Province to determine the aetiology of trunk diseases with reference to trunk diseases occurring on grapevine. Grapevine trunk diseases cause the gradual decline and dieback of vines resulting in a decrease in the vine’s capability to carry and ripen fruit. In recent years, viticulture has been expanding into several of the well established pome fruit growing areas. The presence of trunk pathogens in pome fruit orchards may affect the health of the pome fruit trees as well as cause a threat to young vineyards planted in close proximity to these potential sources of viable inoculum. Several genera containing species known to be involved in trunk disease on pome fruit and grapevine were found, including Diplodia, Neofusicoccum, Eutypa, Phaeoacremonium and Phomopsis. Diplodia seriata and D. pyricolum, were isolated along with N. australe and N. vitifusiforme. Four Phaeoacremonium species, P. aleophilum, P. iranianum, P. mortoniae and P. viticola, two Phomopsis species linked to clades identified in former studies as Phomopsis sp. 1 and Phomopsis sp. 7, and Eutypa lata were found. In addition, Paraconiothyrium brasiliense and Pa. variabile, and an unidentified Pyrenochaetalike species were found. Of these the Phaeoacremonium species have not been found on pear wood and it is a first report of P. aleophilum occurring on apple. This is also a first report of the Phomopsis species and Eutypa lata found occurring on pome trees in South Africa Two new coelomycetous fungi were also found including a Diplodia species, Diplodia pyricolum sp. nov., and a new genus, Pyrenochaetoides gen. nov. with the type species, Pyrenochaetoides mali sp. nov., were described from necrotic pear and apple wood. The combined ITS and EF1-α phylogeny supported the new Diplodia species, which is closely related to D. mutila and D. africana. The new species is characterised by conidia that become pigmented and 1-septate within the pycnidium, and that are intermediate in size between the latter two Diplodia species. Phylogenetic inference of the SSU of the unknown coelomycete provided bootstrap support (100%) for a monophyletic clade unrelated to known genera, and basal to Phoma and its relatives. Morphologically the new genus is characterised by pycnidial with elongated necks that lack setae, cylindrical conidiophores that are seldomly branched at the base, and Phoma-like conidia. The phylogenetic results combined with its dissimilarity from genera allied to Phoma, lead to the conclusion that this species represents a new genus. A pathogenicity trial was undertaken to examine the role of these species on apple, pear and grapevine shoots. N. australe caused the longest lesions on grapevine shoots, while Pyrenochaetoides mali, Pa. variabile, D. seriata and P. mortoniae caused lesions that were significantly longer than the control inoculations. On pears, D. pyricolum and N. australe caused the longest lesions, followed by D. seriata and E. lata. On apples, the longest lesions were caused by N. australe and P. iranianum. D. seriata, D. pyricolum, E. lata, N. vitifusiforme, Pa. brasiliense, P. aleophilum and P. mortoniae also caused lesions on apple that were significantly longer than the control. The study demonstrated that close cultivation of grapevine to apple and pear orchards may have inherent risks in terms of the free availability of viable inoculum of trunk disease pathogens.
No Afrikaans abstract available.

Research and application of microbial control of brown spot disease on the dragon fruit caused by fungi Neoscytalidium dimetiatum have important implications towards the safe and sustainable dragon fruit production. In this article, the research team has identified two strains of microorganisms capable of inhibiting fungi Neoscytalidium dimitiatum denoted A3, B7. Classification results determined that A3 belongs to Actinomyces group 3 with similarities of 100% (1500/1500 bp) with 16S rDNA segment of Streptomyces fradiae; B7 with similarities of 100% (1414/1414 bp) with 16S rDNA segment of bacteria Bacillus polyfermenticus and ensure biosafety when released into the environment
Nghiên cứu ứng dụng vi sinh vật kiểm soát bệnh đốm nâu trên cây thanh long do nấm Neoscytalidium dimetiatum gây ra có ý nghĩa quan trọng hướng tới ngành sản xuất thanh long an toàn và bền vững. Trong bài viết này nhóm nghiên cứu đã xác định được hai chủng vi sinh vật có khả năng ức chế nấm Neoscytalidium dimitiatum cao kí hiệu là A3, B7. Kết quả phân loại xác định chủng A3 thuộc nhóm xạ khuẩn 3 tương đồng 100% (1500/1500 bp) với đoạn 16S rDNA của Streptomyces fradiae; chủng B7 tương đồng 100% (1414/1414 bp) với đoạn 16S của vi khuẩn Bacillus polyfermenticus và đảm bảo an toàn sinh học khi phóng thích ra môi trường

Aquesta tesi s'ha focalitzat en el desenvolupament de noves eines immunoquímiques per al diagnòstic de malalties infeccioses amb l'objectiu d'incrementar l'eficiència dels actuals mètodes de diagnòstic. Concretament, aquesta tesi s'ha centrat en el desenvolupament de tècniques immunoquímiques per a la detecció de Staphylococcus aureus i Pseudomones aeruginosa en mostres biològiques. L'estratègia ha consistit a seleccionar dianes específiques de cadascun dels microorganismes i, mitjançant el disseny racional d'haptens d'immunització, desenvolupar anticossos policlonals específics. Els anticossos han estat avaluats mitjançant assajos de tipus ELISA (enzyme-linked immunosorbent assay) i posteriorment s'han implementat en immunoassajos ELISA en microplaca o de micromatrius (microarrays) fluorescents per a l'anàlisi de mostres biològiques (bacteris en medi de cultiu) i mostres clíniques d'origen respiratori (esput, rentats broncoalveolars i broncoaspirats). Els immunoassajos obtinguts per ambdós bacteris són capaços de detectar les dianes seleccionades amb nivells de detecció útils per a la seva implementació en l'àmbit de diagnòstic, oferint una alternativa prometedora al diagnòstic de malalties causades per aquests bacteris. Com a resultat de la investigació realitzada, s'han generat dues patents (PCT/ES2014/070161 i P201530780), les quals es troben en fase de negociació amb empreses de l'àmbit del diagnòstic. A més s'ha publicat un article de revisió que descriu l'estat de la qüestió pel que fa al potencial de la nanobiotecnologia en el diagnòstic de microorganismes patogen, un article de recerca original, que ja ha estat publicat en la revista Analytica Chimica Acta i que descriu el treball realitzat pel que fa a la detecció immunoquímica de S. aureus, i un tercer article que es troba sota avaluació pels revisors i editors de la revista Analytical Chemistry i que reporta la recerca realitzada pel que fa al desenvolupament d'eines immunoquímiques per al diagnòstic de malalties causades per P. aeruginosa. Per aquest motiu, la tesi s'ha estructurat en format de compendi de publicacions. La tesi també inclou un annex fruit d'una estada predoctoral al grup del Prof. Kim Janda de The Scripps Research Institute (TSRI, CA, EEUU). L'objectiu de l'estada era l'avaluació com a eines terapèutiques dels anticossos produïts en aquesta tesi contra la P. aeruginosa. En aquest sentit, es pretenia investigar el potencial dels anticossos generats per neutralitzar els efectes citotòxics causats per factors de virulència d'aquest microorganisme sobre línies cel•ulars de macròfags. Tot i l'interès d'aquests estudis, aquest treball no es va poder concloure, i no es descarta reprendre'l més endavant.
This thesis has focused on the development of immunochemical new tools for the diagnosis of infectious diseases with the aim of increasing the efficiency of current diagnostic methods. Specifically, this work has focused on the development of immunochemical techniques for the detection of Staphylococcus aureus and Pseudomonas aeruginosa in biological samples. The strategy consisted in selecting specific targets for each of the microorganisms and, specific polyclonal antibodies were developed through rational design of immunization haptens. The antibodies were evaluated by ELISA tests (enzyme-linked immunosorbent assay) and later implemented in microplate ELISA immunoassays and fluorescent microarrays for the analysis of biological samples (bacteria in culture medium) and clinical samples of respiratory origin (sputum and broncoalveolars lavages and broncoaspirats). The immunoassays obtained for both bacteria are capable of detecting the selected targets with detection levels useful for its implementation in the diagnosis field, offering a promising alternative for diagnosing diseases caused by these bacteria. As a result of the research carried out two patents have been generated (PCT/ES2014/070161 and P201530780), which are under negotiation with companies in the field of diagnosis. It has published a review article that describes the state of the art regarding the potential of nanobiotechnology in the diagnosis of pathogenic microorganisms, an original research paper, which was published in the Analytica Chimica Acta journal describing the work regarding the immunochemistry detection of S. aureus, and a third article that is under evaluation by the reviewers and editors of the Analytical Chemistry journal and reporting research conducted regarding the development of immunochemistry tools to diagnose diseases caused by P. aeruginosa.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
Worldwide, the rust diseases cause significant annual wheat yield losses (Wallwork 1992; Chrispeels & Sadava 1994). The utilization of host plant resistance to reduce such losses is of great importance particularly because biological control avoids the negative environmental impact of agricultural chemicals (Dedryver et al. 1996). The wild relatives of wheat are a ready source of genes for resistance to disease and insect pests. A large degree of gene synteny still exists among wheat and its wild relatives (Newbury & Paterson 2003). It is therefore possible to transfer a chromosome segment containing useful genes to a homologous region in the recipient genome without serious disruption of genetic information. Special cytogenetic techniques are employed to transfer genes from the wild relatives to the wheat genomes (Knott 1989). Unfortunately the transfer of useful genes may be accompanied by the simultaneous transfer of undesirable genes or redundant species chromatin which has to be mapped and removed (Feuillet et al. 2007). DNA markers are extremely useful for the characterisation and shortening of introgressed regions containing genes of interest (Ranade et al. 2001), and may also be used for marker aided selection of the resistance when the genes are employed commercially. Eight wheat lines containing translocations/introgressions of wild species-derived resistance genes were developed by the Department of Genetics (SU). These lines are presently being characterized and mapped and attempts are also being made to shorten the respective translocations. This study aimed to find DNA markers for the various translocations and to convert these into more reliable SCAR markers that can be used in continued attempts to characterize and improve the respective resistance sources. A total of 260 RAPD and 21 RGAP primers were used to screen the eight translocations and, with the exception of Lr19, it was possible to identify polymorpic bands associated with each translocation. However, it was not possible to convert all of these into more reliable SCAR markers. The primary reason for this was the low repeatability of most of the bands. Certain marker fragments turned out to be repeatable but could not be converted successfully. Some of the latter can, however, be used directly (in RAPD or RGAP reactions) as markers. The Lr19 translocation used in the study (Lr19-149-299) is a significantly reduced version of the original translocation and failure to identify polymorphisms associated with it can probably be ascribed to its small size. The following numbers of markers (direct and converted into SCARs) were Worldwide, the rust diseases cause significant annual wheat yield losses (Wallwork 1992; Chrispeels & Sadava 1994). The utilization of host plant resistance to reduce such losses is of great importance particularly because biological control avoids the negative environmental impact of agricultural chemicals (Dedryver et al. 1996). The wild relatives of wheat are a ready source of genes for resistance to disease and insect pests. A large degree of gene synteny still exists among wheat and its wild relatives (Newbury & Paterson 2003). It is therefore possible to transfer a chromosome segment containing useful genes to a homologous region in the recipient genome without serious disruption of genetic information. Special cytogenetic techniques are employed to transfer genes from the wild relatives to the wheat genomes (Knott 1989). Unfortunately the transfer of useful genes may be accompanied by the simultaneous transfer of undesirable genes or redundant species chromatin which has to be mapped and removed (Feuillet et al. 2007). DNA markers are extremely useful for the characterisation and shortening of introgressed regions containing genes of interest (Ranade et al. 2001), and may also be used for marker aided selection of the resistance when the genes are employed commercially. Eight wheat lines containing translocations/introgressions of wild species-derived resistance genes were developed by the Department of Genetics (SU). These lines are presently being characterized and mapped and attempts are also being made to shorten the respective translocations. This study aimed to find DNA markers for the various translocations and to convert these into more reliable SCAR markers that can be used in continued attempts to characterize and improve the respective resistance sources. A total of 260 RAPD and 21 RGAP primers were used to screen the eight translocations and, with the exception of Lr19, it was possible to identify polymorpic bands associated with each translocation. However, it was not possible to convert all of these into more reliable SCAR markers. The primary reason for this was the low repeatability of most of the bands. Certain marker fragments turned out to be repeatable but could not be converted successfully. Some of the latter can, however, be used directly (in RAPD or RGAP reactions) as markers. The Lr19 translocation used in the study (Lr19-149-299) is a significantly reduced version of the original translocation and failure to identify polymorphisms associated with it can probably be ascribed to its small size. The following numbers of markers (direct and converted into SCARs) were v identified: S8-introgression (Triticum dicoccoides) = one RAPD and two SCARs; S13-translocation (Aegilops speltoides) = four RAPDs, three RGAPs and five SCARs; S15-translocation (Ae. peregrina) = one RAPD and two SCARs; S20-translocation (Ae. neglecta) = two RAPDs, two RGAPs and one SCAR. The markers are already being employed in current projects aiming to map and shorten these translocations. Some of the markers can be combined in multiplex reactions for more effective mass screening. No repeatable markers could be identified for the four remaining translocations (S12 from Ae. sharonensis; S14 from Ae. kotschyi; Smac from Ae. biuncialis and Lr19-149-299 from Thinopyrum ponticum).

Resumo: Com o objetivo da redução da aplicação de fungicidas na produção de alho, foram estudados os efeitos dos Microrganismos Eficazes (E.M.-4 e E.M.-5) sobre o controle da mancha púrpura, a produção da cultura e as propriedades físicas, químicas e biológicas do solo. Os experimentos foram conduzidos, em condições de campo, por dois e três anos, na Fazenda de Ensino, Pesquisa e Produção da Unesp em São Manuel, SP. O E.M. foi aplicado nos bulbilhos de alho antes do plantio, adicionado ao material orgânico incorporado ao solo e pulverizado nas plantas após incubação ou não com melaço. A utilização do E.M. + melaço (não incubado) não proporcionou controle da doença nos experimentos. No entanto, com a incubação do E.M. + melaço houve redução na severidade da mancha púrpura em um experimento e incremento na emergência e número de folhas verdes por planta em ambos os experimentos. A altura das plantas, superbrotamento, produção, bulbos de maior valor comercial (classes 5+6+7) e as propriedades físicas (densidade do solo, condutividade hidráulica, estabilidade dos agregados, resistência à penetração e infiltração de tinta), químicas e biológicas do solo (conteúdo de polissacarídeos, carbono da biomassa microbiana do solo e atividade da desidrogenase) não foram alteradas pela utilização do E.M. A adição de material orgânico ao solo promoveu maior agregação do solo (estabilidade dos agregados), independentemente dos tratamentos empregados. No entanto, a densidade do solo, a condutividade hidráulica e a resistência à penetração não sofreram alterações com a adição do material orgânico.
Abstract: With the purpose to reduce fungicides application for garlic production, the effects of Effective Microorganisms (E.M.-4 and E.M.-5) were studied on the control of garlic purple blotch, garlic yield and physical, chemical and biological properties of the utilized planting soil. Experiments were carried out under field conditions, for two and three years, an experimental farm, belonging to São Paulo State University, located in São Manuel, São Paulo, Brazil. E.M. was applied on garlic cloves before planting, additioned to the incorporated organic matter and sprayed on garlic aerial part after incubation or not with molasse. When E.M. plus molasse (not incubated) was utilized the control of purple blotch was not observed in two experiments. However, with the incubation of E.M. with molasse, the results showed a reduction of purple blotch severity in one experiment as well as an increment of seedling emergency and number of green leaves per plant in two experiments. Plant height, bulbil sprouting, yield, bulbs with higher market grades (classes 5+6+7) and soil physical (bulk density, hidraulic conductivity, stable soil aggregates, resistance to soil penetration and tint infiltration) chemical and biological (polyssacharides, carbon of microbial biomass and dehydrogenase content) proprieties were not affected by E.M. utilization. Organic matter addition promoted soil agreggation in all treatments, however, soil bulk density, hidraulic conductivity and resistance to soil penetration was not changed by organic matter addition.
Orientador: Chukichi Kurozawa
Coorientador: Roberto Lyra Villas-Bôas
Banca: Chukichi Kurozawa
Banca: Julio Nakagawa
Banca: Rumy Goto
João Batista Vida
Hasime Tokeshi
Doutor

Thesis (MSc (Genetics))--University of Stellenbosch, 2011.
Includes bibliography
ENGLISH ABSTRACT: This study investigated the use of antimicrobial peptides (AMPs) as possible source of resistance against a range of pathogens in grapevine. Whilst the ultimate aim would be to express AMPs in grapevine, the development of transgenic grapevine is time consuming and therefore pre-screening of potential AMPs is necessary. These small molecules, of less than 50 amino acids in length, are expressed by almost all organisms as part of their non-specific defence system. In vitro pre-screening of AMP activity is valuable but is limited since the activity on artificial media may differ from the AMP activity in planta. These tests are also restricted to pathogens which can be cultured in vitro. These limitations can be overcome by using transient expression systems to determine the in planta activity of AMPs against pathogens of interest. In this study transient systems were used to express AMPs in developed plant tissue to test their efficacy against grapevine pathogens such as Agrobacterium vitis, Xylophilus ampelinus and aster yellows phytoplasma. Aster yellows phytoplasma, which was recently discovered in local vineyards, is known to cause extensive damage and therefore pose a great threat to the South African grapevine industry. To study the in planta effect of AMPs against the abovementioned pathogens, transient expression vectors were constructed expressing either of the AMPs D4E1 or Vv-AMP1. D4E1 is a synthetically designed AMP known to be active against bacteria and fungi, while Vv-AMP1, isolated from grapevine berries, has already shown activity against fungi. In a transient approach in grapevine, the expression of foreign genes from viral and non-viral vectors was confirmed by expression of the marker genes β-glucuronidase and Green Fluorescent Protein, while tissue-printing immunoassays confirmed viral replication and systemic spread in Nicotiana benthamiana. The viral vectors were based on the phloem-limited virus grapevine virus A. Only Agrobacterium-mediated 35S transient expression vectors were used for AMP in planta activity screening since the viral-mediated expression in grapevine was insufficient for screening against A. vitis and X. ampelinus as it was restricted to phloem tissues after whole-leaf infiltration. No phytoplasma-infected material could be established and as a result AMP activity screening was only performed against the A. vitis and X. ampelinus. Quantification of the bacteria was performed by qPCR. Vv-AMP1 did not show activity against either of the two bacteria in planta while D4E1 was found to be active against both. The observed in planta activity of D4E1 correlated with the in vitro activity as measured in an AMP plate bioassay. In contrast to in vitro screenings, the in planta AMP activity screening might give a more accurate representation of the potential antimicrobial activity of the peptide in a transgenic plant environment. This study proved that transient expression systems can be used as a pre-screening method of AMP activity in planta against grapevine pathogens, allowing the screening of various AMPs in a relatively short period of time before committing to transgenic grapevine development.
AFRIKAANSE OPSOMMING: Hierdie studie het die gebruik van antimikrobiese peptiede (AMPe) as 'n moontlik bron van weerstand teen 'n reeks van patogene in wingerd ondersoek. Alhoewel die uiteindelike doel sal wees om AMPe uit te druk in wingerd, is transgeniese wingerd ontwikkeling tydrowend en daarom is vooraf evaluering van potensiële AMPe nodig. Hierdie klein molekules, van minder as 50 aminosure in lengte, word uitgedruk deur amper alle organismes as deel van hul nie-spesifieke verdedigingsisteem. In vitro vooraf evaluering van AMP aktiwiteit is van waarde, maar is beperk aangesien die aktiwiteit op kunsmatige media mag verskil van die AMP-aktiwiteit in planta. Hierdie toetse is ook beperk tot patogene wat in vitro gekweek kan word. Hierdie beperkinge kan oorkom word deur gebruik te maak van tydelike uitdrukkingsisteme om die in planta aktiwiteit van AMPe te bepaal teen patogene van belang. In hierdie studie is tydelike uitdrukkingsisteme gebruik om AMPe uit te druk in ontwikkelde plantweefsel om hul effektiwiteite te toets teen wingerdpatogene soos Agrobacterium vitis, Xylophilus ampelinus en aster yellows fitoplasma. Aster yellows fitoplasmas, wat onlangs in plaaslike wingerde ontdek is, is bekend vir die uitgebreide skade wat hul aanrig en hou daarom 'n groot bedreiging in vir die Suid-Afrikaanse wingerd industrie. Om die in planta effek van AMPe teen die bogenoemde patogene te bestudeer is tydelike uitdrukkingsvektore ontwikkel wat die AMPe D4E1 of Vv-AMP1 uitdruk. D4E1 is 'n sinteties-ontwerpte AMP wat aktief is teen bakterieë en fungi, terwyl Vv-AMP1, wat uit druiwekorrels geïsoleer is, alreeds aktiwiteit teen fungi getoon het. In 'n tydelike uitdrukkingsbenadering in wingerd is die uitdrukking van transgene, vanaf virus of nie-virus gebaseerde vektore, bevestig deur die uitdrukking van die merker gene β-glukuronidase en die Groen Fluoresserende Proteïen, terwyl weefsel afdrukkings-immunotoetse virus replisering en sistemiese beweging in Nicotiana benthamiana bevestig het. Die virusvektore was gebaseer op die floëem-beperkte virus, wingerdvirus A. Slegs Agrobacterium-bemiddelde 35S tydelike uitdrukkingsvektore is gebruik om die AMP in planta aktiwiteit te bepaal aangesien die virus-bemiddelde uitdrukking in wingerd onvoldoende was vir evaluering teen A. vitis en X. ampelinus weens die beperking tot die floëem weefsel na infiltrering van die totale blaar. Geen fitoplasma geïnfekteerde materiaal kon gevestig word nie, en daarom is AMP aktiwiteitsevaluering slegs teen A. vitis en X. ampelinus uitgevoer. Kwantifisering van die bakterieë is deur middel van qPCR uitgevoer. Vv-AMP1 het geen aktiwiteit getoon teen enige van die bakterieë in planta nie, terwyl D4E1 aktief was teen beide. Die waargenome in planta aktiwiteit van D4E1 het ooreengestem met die in vitro aktiwiteit soos bepaal deur 'n AMP plaat bio-toets. In kontras tot in vitro evaluering kan die in planta AMP-aktiwiteit evaluering 'n meer akkurate voorspelling bied van die potensiële antimikrobiese aktiwiteite van die peptied in 'n transgeniese plant omgewing. Hierdie studie het bewys dat tydelike uitdrukkingsisteme gebruik kan word as 'n voorafgaande evalueringsmetode vir AMP in planta aktiwiteit teen wingerdpatogene, wat die evaluering van 'n verskeidenheid AMPe in 'n relatiewe kort tydperk toelaat voor verbintenis tot die ontwikkeling van transgeniese wingerd.

Dissertation (PhD)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: Plants have developed sophisticated means of combating plant diseases. The events that prepare the plant for, and follow plant-pathogenic interactions, are extremely complex and have been the topic of intensive investigation in recent years. These interactions involve a plethora of genes and proteins, and intricate regulation thereof; from the host and pathogen alike. Studying the contribution of single genes and their encoded proteins to the molecular dialogue between plant and pathogen has been a focus of plant molecular biologists. To this end, a gene encoding a polygalacturonase-inhibiting protein (PGIP) was recently cloned from Vitis vinifera. These proteins have the ability to inhibit fungal endopolygalacturonases (ePGs), enzymes which have been shown to be required for the full virulence of several fungi on their respective plant hosts. The activity of PGIP in inhibiting fungal macerating enzymes is particularly attractive for the improvement of disease tolerance of crop species. The VvPGIP-encoding gene was subsequently transferred to Nicotiana tabacum for high-level expression of VvPGIP. These transgenic plants were found to be less susceptible to infection by Botrytis cinerea in an initial detached leaf assay. Also, it was shown that ePG inhibition by protein extracts from these lines correlated to the observed decrease in susceptibility to B. cinerea. This study expands on previous findings by corroborating the antifungal nature of the introduced PGIP by whole-plant, timecourse infection assays. Six transgenic tobacco lines and an untransformed wildtype (WT) were infected and the lesions measured daily from day three to seven, and again at day 15. The transgenic lines exhibited smaller lesions sizes from three to seven days post-inoculation, although these differences only became statistically significant following seven days of incubation. At this point, four of the six lines exhibited significantly smaller lesions than the WT, with reductions in disease susceptibility ranging between 46 and 69% as compared to the WT. Two of the lines exhibited disease susceptibility comparable to the WT. In these resistant plant lines, a correlation could be drawn between Vvpgip1 expression, PGIP activity and ePG inhibition. These lines were therefore considered to be PGIP-specific resistant lines, and provided ideal resources to further study the possible in planta roles of PGIP in plant defense. The current hypothesis regarding the role(s) of PGIP in plant defense is twofold. Firstly, PGIPs have the ability to specifically and effectively inhibit fungal ePGs. This direct inhibition results in reduced fungal pathogenicity. Alternatively, unhindered action of these enzymes results in maceration of plant tissue and ultimately, tissue necrosis. Subsequently, it could be shown that, in vitro, the inhibition of ePGs prolongs the existence of oligogalacturonides, molecules with the ability to activate plant defense responses. Thus, PGIPs limit tissue damage by inhibition of ePG; this inhibition results in activation of plant defense responses aimed at limiting pathogen ingress. Several publications reported reduced susceptibility to Botrytis in transgenic plant lines overexpressing PGIP-encoding genes. However, none of these publications could expand on the current hypotheses regarding the possible in planta roles of PGIP in plant defense. In this study we used transgenic tobacco lines overexpressing Vvpgip1 as resources to study the in planta roles for PGIP. Transcriptomic and hormonal analyses were performed on these lines and a WT line, both before and following inoculation with Botrytis cinerea. Transcriptomic analysis was performed on uninfected as well as infected tobacco leaf material utilizing a Solanum tuberosum microarray. From the analysis with healthy, uninfected plant material, it became clear that genes involved in cell wall metabolism were differentially expressed between the transgenic lines and the WT. Under these conditions, it could be shown and confirmed that the gene encoding tobacco xyloglucan endotransglycosylase (XET/XTH) was downregulated in the transgenic lines. Additionally, genes involved in the lignin biosynthetic pathway were affected in the individual transgenic lines. Biochemical evidence corroborated the indication of increased lignin deposition in their cell walls. Additionally, phytohormone profiling revealed an increased indole-acetic acid content in the transgenic lines. These results show that constitutive levels of PGIP may affect cell wall metabolism in the Vvpgip1-transgenic lines which may have a positive impact on the observed reduced susceptibilities of these plants. An additional role for PGIP in the contribution to plant defenses is therefore proposed. PGIP may directly influence defense responses in the plant leading to the strengthening of cell walls. This might occur by virtue of its structural features or its integration in the cell wall. These reinforced cell walls are thus “primed” before pathogen ingress and contribute to the decrease in disease susceptibility observed in lines accumulating high levels of PGIP. Transcriptional and hormonal analyses, at the localized response, were performed on Botrytis-infected leaf tissue of the transgenic lines and a WT line. Several Botrytis responsive genes were found to be upregulated in both the WT and the transgenic lines. Although limited differential expression was observed between the two genotypes, the analyses identified a gene which was upregulated two-fold in the transgenic lines, as compared to WT. This was confirmed by quantitative Real-Time PCR. This gene is involved in the lipoxygenase pathway, specifically the 9-LOX branch, leading to the synthesis of the divinyl ether oxylipins colneleic and colnelenic acid, which show inhibitory effects on Botrytis spore germination. Phytohormone profiling revealed that the transgenic lines accumulated more of the defense-related hormone pool of jasmonates. These are formed via the 13-LOX pathway and have been shown to be important for the restriction of Botrytis growth at the site of infection. Collectively, the results from the infection analyses indicate that in these transgenic lines, both branches of the lipoxygenase pathway are differentially induced at the level of the localized response to Botrytis infection. Similarly, an increased induction of the synthesis of the defense-related hormone salicylic acid could be observed, although this hormone did not accumulate to significantly higher levels. These results are the first report of differential induction of a defense-related pathway in pgip-overexpressing lines and substantiate the proposal that following ePG inhibition by PGIP, signaling which activates plant defense responses, takes place. Taken together, these results significantly contribute to our understanding of the in planta role of PGIP in plant defense responses.
AFRIKAANSE OPSOMMING: Plante het deur evolusie gesofistikeerde meganismes teen die aanslag van plantsiektes ontwikkel. Die gebeure wat die plant voorberei, asook dié wat op plant-patogeen interaksies volg, is uiters kompleks en vorm die kern van verskeie navorsingstemas die afgelope paar jaar. Etlike plant- én patogeengene en proteïene is by hierdie interaksies betrokke en aan komplekse reguleringsprosesse onderworpe. Die bestudering van die bydrae van enkelgene en hul gekodeerde proteïene tot die molekulêre interaksie tussen ‘n plant en patogeen is ‘n sterk fokus van plant-molekulêre bioloë. Met hierdie doel as fokus, is ‘n geen wat vir ‘n poligalakturonaseinhiberende proteïen (PGIP) kodeer, van Vitis vinifera gekloneer. Hierdie proteïene beskik oor die vermoë om fungiese endopoligalakturonases (ePG's), ensieme wat benodig word vir die virulensie van verskeie fungi op hul gasheerplante, te inhibeer. Die inhibisie van ePG's deur PGIP en die gepaardgaande verminderde weefseldegradasie is ‘n baie belowende strategie vir die verbetering van verboude gewasse se patogeentoleransie. Die VvPGIPenkoderende geen is gevolglik na Nicotiana tabacum oorgedra vir hoëvlakuitdrukking van VvPGIP. Daar is gevind dat hierdie transgeniese plante minder vatbaar vir Botrytis cinerea-infeksies was in ‘n inisiële antifungiese toets wat gebruik gemaak het van blaarweefsel wat van die moederplant verwyder is. Daar is ook ‘n korrelasie gevind tussen B. cinerea-siekteweerstand en ePG-inhibisie deur proteïenekstrakte van die transgeniese populasie. Die huidige studie bou voort op en bevestig vorige bevindinge betreffende die antfungiese aard van die heteroloë PGIP in die heelplant en oor tyd. Ses transgeniese tabaklyne en 'n ongetransformeerde wilde-tipe (WT) is geïnfekteer en die lesies is vanaf dag drie tot sewe, en weer op dag 15, gemeet. Die transgeniese lyne het in die tydperk van drie tot sewe dae ná-inokulasie kleiner lesies as die WT getoon, alhoewel hierdie verskille slegs statisties beduidend geword het na sewe dae van inkubasie. Op daardie tydstip het vier van die ses lyne aansienlik kleiner lesies as die WT getoon, en verlagings in siektevatbaarheid het, in vergelyking met die WT, van 46% tot 69% gewissel. Twee van die lyne het siektevatbaarheid getoon wat vergelykbaar was met dié van die WT. In die siekteweerstandbiedende plantlyne was daar 'n verband tussen Vvpgip1-ekspressie, PGIP-aktiwiteit en ePG-inhibisie. Hierdie plantlyne is dus as PGIP-spesifieke siekteweerstandslyne beskou en dien dus as ideale eksperimentele bronne vir die ontleding van die moontlike in plantafunksies van PGIP in plantsiekteweerstandbiedendheid. Die huidige hipotese betreffende die funksie(s) van PGIP in plantsiekteweerstand is tweeledig. Eerstens het PGIP die vermoë om fungusePG's spesifiek en doeltreffend te inhibeer. Hierdie direkte inhibisie veroorsaak ‘n vermindering in patogenisiteit van die fungus op die gasheer. Indien ePG's egter hulle ensimatiese aksie onverstoord voortsit, sal weefseldegradasie en uiteindelik weefselnekrose die gevolg wees. Daar kon ook bewys word dat die in vitroinhibisie van ePG's deur PGIP die leeftyd van oligogalakturoniede, molekules wat die vermoë het om die plantweerstandsrespons aan te skakel, kan verleng. PGIP het dus nie net die vermoë om ePG's, en dus weefseldegradasie, te inhibeer nie; maar hierdie inhibisie lei ook daartoe dat plantweerstandsresponse aangeskakel word met die oog op die vermindering van patogeenindringing. Verskeie publikasies het reeds gerapporteer oor verminderde Botrytisvatbaarheid in PGIP transgeniese plantlyne. Geeneen van hierdie publikasies kon egter uitbrei op die huidige hipotese aangaande die moontlike in planta-funksie van PGIP in plantsiekteweerstand nie. In hierdie studie is transgeniese tabaklyne wat PGIP ooruitgedruk gebruik om hierdie moontlike in planta-funksies vir PGIP uit te klaar. Transkriptoom- en hormonale analises is op hierdie plantlyne en ‘n WT voor en ná inokulasie met die nekrotroof Botrytis cinerea uitgevoer,. Transkriptoomanalises is uitgevoer op ongeïnfekteerde, sowel as geïnfekteerde tabakblaarmateriaal deur gebruik te maak van ‘n Solanum tuberosum-mikroraster. Die analises met gesonde, ongeïnfekteerde plantmateriaal het daarop gewys dat gene betrokke by selwandmetabolisme tussen die transgeniese lyne en die WT verskillend uitgedruk was. Dit kon bewys word dat, sonder infeksiedruk, die geen wat xiloglukaan-endotransglikosilase (XET) kodeer, in die transgeniese lyne afgereguleer was. Gene wat betrokke is in die lignien-biosintetiese pad was ook in die individuele transgeniese lyne beïnvloed. Biochemiese toetse het ook die aanduiding van verhoogde ligniendeposisie in die transgeniese lyne se selwande bevestig. Addisionele fitohormoonprofiele het getoon dat hierdie lyne ook beskik oor verhoogde vlakke van indoolasynsuur (IAA). Hierdie resultate wys daarop dat konstitutiewe vlakke van PGIP selwandmetabolisme in die Vvpgip1-transgeniese lyne moontlik kan beïnvloed, wat plantsiekteweerstand in dié lyne positief kan beïnvloed. Dit wil dus voorkom asof PGIP 'n bykomende funksie in plantsiekteweerstand het. Plantweerstandsreponse kan direk deur PGIP beïnvloed word, wat tot die versterking van plantselwande kan lei; dit kan geskied by wyse van die strukturele eienskappe van die proteïen of die integrasie daarvan in die selwand. Hierdie selwande is dus “voorberei” alvorens patogeenindringing plaasvind en kon bydra tot die verminderde siektevatbaarheid wat waargeneem is in lyne wat hoë vlakke van PGIP akkumuleer. Transkriptoom- en hormonale analises is ook uitgevoer op Botrytisgeïnfekteerde blaarmateriaal van beide die transgeniese lyne en ‘n WT. Verskeie Botrytis-responsgene is in beide die transgeniese lyne en die WT opgereguleer. Differensïele geenekspressie tussen die twee genotipes was taamlik beperk, maar in die analises kon ‘n geen geïdentifiseer word wat tweevoudig in die transgeniese lyne opgereguleer was in vergelyking met die WT. Hierdie resultaat is ook bevestig met behulp van die “Real-Time” Polimerasekettingreaksie (PKR). Hierdie geen is betrokke in die lipoksigenase (LOX) -pad (spesifiek die 9-LOXarm), wat tot die sintese van die diviniel-eter oksilipiene “colneleic-” en “colnelenic”-suur lei. Daar is al bewys dat hierdie twee verbindings Botrytisspoorontkieming kan inhibeer. Fitohormoonprofiele van die geïnfekteerde plante het gewys dat die transgeniese lyne verhoogde vlakke van die poel van jasmonate wat plantsiekteweerstands-hormone is, ná inokulasie akkumuleer. Hierdie hormone word in die 13-LOX-arm van die lipoksigenase pad gevorm en is belangrik vir die beperking van Botrytis by die infeksiesetel. Die resultate van die analises wat op Botrytis-infeksie volg, dui daarop dat beide arms van die lipoksigenasepad in die transgeniese lyne verskillend by die lokale respons geïnduseer word. ‘n Verhoogde induksie van ‘n ander plantsiekteweerstandshormoon, salisielsuur, kon ook opgemerk word, alhoewel die totaal geakkumuleerde vlakke nie beduidend hoër was as dié van die WT nie. Hierdie resultate is die eerste wat onderskeidende induksie van ‘n siekteweerstandspad in enige van die pgip-ooruitgedrukte plantlyne rapporteer. Daarmee ondersteun dit ook die hipotese dat, seintransduksie wat plantweerstandsresponse aanskakel, ná inhibisie van ePG deur PGIP plaasvind. Die resultate wat met hierdie studie verkry is, dra dus beduidend by tot die huidige kennis van die in planta-funksie van PGIP in plantsiekteweerstandsresponse.

The bacterium Chlamydia trachomatis as well as the Human Immunodeficiency virus-1 (HIV-1) are sexually transmitted pathogens; both can infect monocytes/ macrophages and have an obligate intracellular replication cycle. It has been hypothesized that sexually transmitted infections (STIs), including genital Chlamydia, enhance HIV-1 transmission; yet, the underlying biological mechanisms remain unclear. The pathogen¡¯s common biology makes it possible that C. trachomatis and HIV mutually affect each other¡¯s replication cycle during monocyte/macrophage co-infection. To test this hypothesis we used two different bacteria strains in an in vitro HIV co-infection model: Latently virus-infected U1 promonocytes were either inoculated with C. trachomatis serovar D or serovar L2. It was found that serovar D, but not the LGV serovar L2, quickly and significantly reactivates integrated HIV-1 provirus. This reactivation depends on viable bacteria but seemingly neither on a microbial pathogen-associated molecular pattern (PAMP) nor on de novo gene expression by C. trachomatis. Hence, it could be triggered by a preformed protein that the bacterium translocates into the human cell. Because virus production occurs in Chlamydia-infected as well as Chlamydia-free cells and cannot be induced by conditioned culture medium, we propose a cell contact-dependent signal to U1 bystander cells. The HIV reactivation mechanism involves NF-kappaB activation but is independent of eukaryotic de novo protein translation. In summary, the findings might describe a novel Chlamydia-mediated HIV-1 reactivation mechanism from latently infected cells.

Os pacientes com Síndrome de Down (SD) possuem grande incidência de doença periodontal (DP), caracterizada por um curso precoce e com maior severidade. O estudo de metaboloma pode contribuir para o entendimento deste curso da doença, identificando possíveis metabólitos como biomarcadores nestes indivíduos. Para entender o perfil metabolômico dos indivíduos com síndrome de Down e a sua relação com a doença periodontal, realizamos a identificação de metabólitos salivares de adolescentes e adultos jovens, entre 12 e 21 anos, ambos os gêneros. Foram coletados dados sobre o estado geral de saúde e realizados exames clínicos bucais, como índice de higiene oral simplificado, sangramento e profundidade de sondagem. Para a análise do metaboloma foi coletada amostra de saliva não estimulada, analisadas por meio de cromatografia gasosa acoplada á espectrometria de massas. Saliva e fluido crevicular gengival também foram coletados para identificação microbiana através do MALDI-TOF. Os dados encontrados foram submetidos a análise estátisca por meio da Análise dos Componentes Principais (PCA) e quantificação relativa dos metabólitos foi avaliada por testes não paramétricos, Mann-Whitney e Kruskal-Wallis. Foi possível observar através dos modelos de PCA separação dos indivíduos com SD e controles, independente da doença periodontal. A quantificação relativa revelou maiores níveis de glicina, lprolina, l-leucina, l-serina, ácido palmítico, ácido pentanóico, ácido tetradecanóico, tirosina e l-fenilalanina nos grupos SD quando comparados aos controles. Controles com DP também apresentaram níveis elevados de glicina, l-alanina, l-serina e manopiranose quando comparados com controles saudáveis. A microbiota de indivíduos com SD apresentous diferenças siginificantes em relação aos individuos controles, principalmente para Rothia dentocariosa, Staphylococcus epidermidis, Tannerella forsythia quando avaliado a saliva e A. Actinomycetemcomitans, Micrococcus luteus, Rothia aeria, Treponema denticola no fluido crevicular gengival. Em conclusão, o perfil metabolômico impresso nos indivíduos com SD difere significativamente dos indivíduos controles, independente da doença periodontal. Entretanto, os metabólitos que diferenciam indivíduos controles com e sem DP, apresentam-se elevados em todos indivíduos com SD, promovendo novos \"insights\" para o perfil metabólico relacionado a DP na SD.
Down Syndrome (DS) patients have a high incidence of periodontal disease (PD), characterized by an early course and greater severity. The metabolome study may contribute to the understanding of the disease course, identifying possible metabolites as biomarkers in these individuals. To understand the metabolomic profile of the DS and their relationship with PD, we conducted the identification of salivary metabolites of adolescents and young adults between 12 and 21 years, both genders. Data were collected on general health and was performed oral clinical examination, as the IHOS, bleeding index and probing depth. For metabolome analysis was collected unstimulated saliva sample, analyzed by gas chromatography coupled to mass spectrometry. Saliva and gingival crevicular fluid were also collected for microbial identification by MALDI-TOF. Data were submitted to analysis-statistic by PCA and relative quantification of metabolites was evaluated by Mann-Whitney and Kruskal-Wallis tests. It can be observed through the PCA models separation of DS groups and controls groups, regardless of periodontal disease. Relative quantification showed higher levels of glycine, L-proline, L-leucine, L-serine, palmitic acid, pentanoic acid, tetradecanoic acid, tyrosine and L-phenylalanine in the SD groups when compared to controls groups. Controls with PD also showed high levels of glycine, L-alanine, L-serine and mannopyranose compared with healthy controls. The microbiota of individuals with DS groups show significant differences compared to control groups, especially for Rothia dentocariosa, Staphylococcus epidermidis, Tannerella forsythia when evaluated saliva and A. actinomycetemcomitans, Micrococcus luteus, Rothia aeria, Treponema denticola in gingival crevicular fluid. In conclusion, the printed metabolomic profile in individuals with Down syndrome differs significantly from control subjects, regardless of periodontal disease. However, the metabolites that distinguish controls group with and without PD, show up high in all DS individuals, promoting new \"insights\" to the metabolic profile related to PD in DS.

Phytophthora cinnamomi has been recognised as a key threatening process to Australia's biodiversity by the Commonwealth's Environment Protection and Biodiversity Conservation Act 1999. Despite over 80 years of extensive research, its exact mode of survival is still poorly understood. It is widely accepted that thin- and thick-walled chlamydospores are the main survival propagules while oospores are assumed to play no role in the survival of the pathogen in the Australian environment, yet evidence is limited. The saprophytic ability of the pathogen is still unresolved despite the important role this could play in the ability of the pathogen to survive in the absence of susceptible hosts. This thesis aimed to investigate chlamydospores, oospores and the saprophytic ability of P. cinnamomi to determine their contribution to survival. Phytophthora cinnamomi did not show saprophytic ability in non-sterile soils. The production of thick-walled chlamydospores and selfed oospores of P. cinnamomi in vitro was documented. Thick-walled chlamydospores were sporadically formed under sterile and non-sterile conditions in vitro but exact conditions for stimulating their formation could not be determined. The formation of thick-walled chlamydospores emerging from mycelium of similar wall thickness was observed, challenging the current knowledge of chlamydospore formation. Selfed oospores were abundant in vitro on modified Ribeiro's minimal medium in one isolate. Three other isolates tested also produced oospores but not in large numbers. Although the selfed oospores did not germinate on a range of media, at least 16 % were found to be viable using Thiozolyl Blue Tetrazolium Bromide staining and staining of the nuclei with 4', 6-diamidino-2-phenylindole.2HCl (DAPI). This indicated the potential of selfed oospores as survival structures and their ability to exist dormantly. The ability of phosphite to kill chlamydospores and selfed oospores was studied in vitro. Results challenged the efficacy of this chemical and revealed the necessity for further study of its effect on survival propagules of P. cinnamomi in the natural environment. Phosphite was shown to induce dormancy in thin-walled chlamydospores if present during their formation in vitro. Interestingly, dormancy was only induced by phosphite in isolates previously reported as sensitive to phosphite and not those reported as tolerant. Chlamydospores were produced uniformly across the radius of the colony on control modified Ribeiro's minimal medium but on medium containing phosphite (40 or 100 mcg ml-1), chlamydospore production was initially inhibited before being stimulated during the log phase of growth. This corresponded to a point in the colony morphology where mycelial density changed from tightly packed mycelium to sparse on medium containing phosphite. This change in morphology did not occur when the pathogen was grown on liquid media refreshed every four days, and chlamydospores were evenly distributed across the radius of these colonies. This trend was not observed in selfed oospores produced in the presence of phosphite. Selfed oospore production was found to be inhibited by phosphite at the same concentrations that stimulated chlamydospore production. Isolates of P. cinnamomi were transformed using a protoplast/ polyethylene glycol method to contain the Green Fluorescent Protein and geneticin resistance genes to aid in future studies on survival properties of the organism. Although time constraints meant the stability of the transgene could not be determined, it was effective in differentiating propagules of the transformed P. cinnamomi from spores of other microrganisms in a non-sterile environment. Two different sized chlamydospores (approximately 30 mcg diameter and < 20 mcg diameter) were observed in preliminary trials of transformed P. cinnamomi inoculated lupin roots floated in non-sterile soil extracts and these were easily distinguished from microbial propagules of other species. The growth and pathogenicity was reduced in two putative transformants and their ability to fluoresce declined over ten subcultures but they still remained resistant to geneticin. This study has improved our knowledge on the survival abilities of P. cinnamomi in vitro and has provided a useful tool for studying these abilities under more natural glasshouse conditions. Important implications of phosphite as a control have been raised.

Thesis (M. Tech.) -- Central University of Technology, Free State, 2010
South Africa currently faces one of the highest HIV prevalence rates in the world. As this prevalence rises, the strain placed on its hospitals is likely to increase due to the shortage of beds. The devastating effects of HIV/AIDS initiated the establishment of a hospice which is a non-governmental organisation whose goal is the provision of care for terminally ill patients, either in their homes, in hospitals or in a hospice’s own in-patients wards. Part of the hospice’s mission is to offer palliative care without charge to anyone who requires it. The basic elements of hospice care include pain and symptom management, provision of support to the bereaving family and promoting a peaceful and dignified death. This also includes the provision of cooked foods to the patients using the kitchen facilities of the hospices for this activity. It is well known that the kitchen is particularly important in the spread of infectious disease in the domestic environment due to many activities that occur in this particular setting. Food and water safety is especially important to the persons infected with the human immunodeficiency virus (HIV) or with immunodeficiency syndrome (AIDS).It is estimated that food-borne pathogens (disease–causing agents) are responsible for 76 million illnesses, some resulting in death, in the United States alone every year. In one study of patients with AIDS, two-thirds had diarrhoeal disease and in two-thirds of these, the following enteric pathogens were identified: Salmonella, Shigella, Listeria, Yersnia, Cryptosporidium, Entamoeba histolylica and Campylobacter sp. In an epidemiological study of patients with HIV infection a close association was found between consumption of raw or partially cooked fish and antimicrobial-resistant Mycobacterium avium complex. Antibiotic resistance in food-borne pathogens has become a reality and this poses a serious threat to the medical fraternity since it diminishes the effectiveness of treatment. This study was undertaken to determine the prevalence of foodborne pathogens including bio aerosols isolated from the kitchen surfaces and food handler’s before and after cooking. The antibiotic resistance of the isolated pathogens was further determined to assess their impact on treatment. The following microbiota were isolated: Total viable counts (TVC), Coliforms, Escherichia coli, Staphylococcus aureus, Pseudomonas and presumptive Salmonella. The hospices had high counts of E.coli and S.aureus on the cutting boards for the breakfast session compared to the traditional home based kitchens. It was speculated that this could have originated from crosscontamination via the foodhandler’s hands and the food served. It is evident from the results that hospices lack a management system regarding the prevalence of E. coli as it was present on the cutting boards throughout the food preparation sessions. Gram negative organisms (coliform and P. aeruginosa) were in particular both resistant to oxacillin and this pose a great challenge in this particular setting. This can be addressed by putting emphasis on hygiene as a strategy per se for reducing antibiotic resistance.

Metagenomics is the study of the collective genomes of the members of a microbial community. In contrast to conventional culture methods, sequence-based metagenomics exclusively relies on sequence analysis without culturing microorganisms. This approach has revolutionised our understanding of the complexity of microbiomes. As such, microbial profiling, particularly of microbiomes in humans that appear to play key roles in numerous disease phenotype, may provide information to help define associated underlying aetiological mechanisms. However, a number of available metagenomic approaches have different biases in the identification and quantification of the microbial composition, resulting in misinterpretation of the accrued data which subsequently affect conclusions. Therefore, the aim of this study was to interrogate sources of error in various methodologies in sequence-based metagenomic analysis. In this study, genomic DNA of common bacterial pathogens representative of both Gram-positive and Gram-negative organisms mixed at defined quantitative proportions were used as a standardised metagenomics control material (MCM) in order to assess the comparative accuracy of different approaches, i.e. 16S ribosomal RNA (rRNA) profiling and metagenomic shot~un-sequencing. Sources of bias in 16S rRNA including primer-template mismatches, primer design, and bioinformatics analytical tools were identified. Whole genome sequencing generated a high precision of microbial profiling. Bias was also observed due to DNA extraction protocol when the whole cell material (WCM) containing a bacteriologically quantitated range of bacteria was used. This study also suggested that the MCM provided the opportunity to develop species specific assays to detect multiple bacterial pathogens collected from the clinical samples by using high-throughput quantitative PCR (ht-qPCR). In conclusion, the methodology applied to microbial profiling analysis must consider sources of error and methods of standardisation such as those described here. Moreover, ht-qPCR demonstrated the value of a high-throughput bacterial detection technique for clinical diagnostic applications. This thesis has thus applied the principles of metrology to generate, characterise and evaluate whole organism and DNA based quantitative control materials as an essential pre-requisite for the precise and accurate biological interpretation of both 16S profiling and metagenomic analysis of human diseases.

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Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Thesis (MScAgric )--Stellenbosch University, 2004.
ENGLISH ABSTRACT: In an attempt to combat some of the pathogens that are associated with trunk diseases and disorders of grapevines, research in this thesis focused on the taxonomy and pathological aspects of Coniellai/Pilidiella, Botryosphaeria and Phomopsis spp. Previously, conidial pigmentation was used to separate Pilidiella from Coniella. Recently, however, the two genera have been regarded as synonymous, with the older name, Coniella, having priority. The most important species in the Coniellai/Pilidiella complex of grapevines is C. diplodiella (Speg.) Petr. & Syd., the causal organism of white rot of grapevines. Previous studies found it difficult to distinguish between C. diplodiella and C. fragariae (Oudem.) B. Sutton, which is known to occur in soil and caused leaf diseases of Fragaria and Eucalyptus. Both these species have previously been reported from South Africa. None of the reports on C. diplodiella could be scientifically substantiated; therefore it is still a quarantine organism. However, this status has been questioned. Based on sequence analyses of the internal transcribed spacer region (ITS 1, ITS 2), 5.8S gene, large subunit (LSU) and elongation factor 1- α gene (EF l- α) from the type species of Pilidiella and Coniella, Coniella was separated from Pilidiella, with the majority of taxa residing in Pilidiella. Pilidiella is characterised by species with hyaline to pale brown conidia (avg. length: width >1.5), with Coniella having dark brown conidia (avg. length: width ≤1.5). Pilidiella diplodiella, previously C. diplodiella, causal organism of white rot of grapevines, was shown to be an older name for C. petrakii. This fungus is present in South Africa and is therefore no longer of quarantine importance. Based on analyses of the histone (H3) gene sequences of isolates in the P. diplodiella species complex, P. diplodiella was separated from a newly described species, P. diplodiopsis. A new species, P. eucalyptorum, is proposed for isolates formerly treated as C. fragariae, associated with leaf spots of Eucalyptus spp. This species clustered basal to Pilidiella, and may represent yet a third genus within this complex. Pilidiella destruens was newly described as anamorph of Schizoparme destruens, which is associated with twig dieback of Eucalyptus spp. in Hawaii. The genus Botryosphaeria Ces. & De Not. are known to be cosmopolitan, with broad host ranges and geographical distributions. Several saprotrophic species have been reported from grapevines, while others are severe pathogens of this host. These species include B. dothidea (Moug.: Fr.) Ces. & De Not., B. parva Pennycook & Samuels, B. obtusa (Schwein.) Shoemaker, B. stevensii Shoemaker, B. lutea A.J.L. Phillips and B. ribis Grossenb. & Duggar. Species reported from South Africa as grapevine pathogens are B. obtusa, B. dothidea, B. ribis and B. vitis (Schulzer) Sacco. In the present study, morphological, DNA sequence data (ITS 1, 5.8S, ITS 2 and EFI-α) and pathological data were used to distinguish 11 Botryosphaeria spp. associated with grapevines from South Africa and other parts of the world. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina and a Diplodia sp. were confirmed from grapevines in South Africa, while Diplodia porosum, Fusicoccum viticlavatum and F. vitifusiforme were described as new species. Although isolates of B. dothidea and B. stevensii were confirmed from grapevines in Portugal, neither of these species, nor B. ribis, were isolated in this study. All grapevine isolates from Portugal, formerly presumed to be B. rib is, are identified as B. parva based on EF1-α sequence data. Artificial inoculations on grapevine shoots showed that B. australis, B. parva, B. ribis and B. stevensii are more virulent than the other species studied. The Diplodia sp. collected from grapevine canes was identified as morphologically similar, but phylogenetically distinct from D. sarmentorum, while D. sarmentorum was confirmed as anamorph of Otthia spiraeae, the type species of the genus Otthia (Botryosphaeriaceae). A culture identified as O. spiraeae clustered within Botryosphaeria, and is thus regarded as a probable synonym. These findings confirm earlier suggestions that the generic concept of Botryosphaeria should be expanded to include genera with septate ascospores and Diplodia anamorphs. The genus Phomopsis (Sacc.) Bubak contains many species that are plant pathogenic or saprotrophic. Ten species are known from grapevines. However, only two have been confirmed as being pathogenic, namely P. viticola (Sacc.) Sacc., causal organism of Phomopsis cane and leaf spot and P. vitimegaspora Kuo & Leu (teleomorph Diaporthe kyushuensis Kajitani & Kanem.), causal organism of swelling arm disease of grapevines. P. amygdali (Delacr.) 1.1. Tuset & M.T. Portilla, a known pathogen from Prunus sp., was shown to be a possible pathogen of grapevines in a previous study. D. perjuncta Niessl. causes bleaching of dormant canes only and is therefore of little importance as a grapevine pathogen. Recently a number of Phomopsis isolates were obtained from grapevines in the Western Cape province of South Africa. Isolations were made from Phomopsis-like symptoms, pruning wounds and asymptomatic nursery plants. These isolates showed great variation in morphology and cultural characteristics. Earlier taxonomic treatments of Phomopsis, based species identification on host specificity, cultural characteristics and morphology. Recent studies have indicated that these characteristics can no longer be used to distinguish species of Phomopsis due to wide host ranges and morphological plasticity of some species. The use of anamorph/teleomorph relationships in species identification is also untenable, since Diaporthe teleomorphs have only been described for approximately 20% of the known Phomopsis species. In this study morphological data, DNA sequences (ITS-I, 5.8S, ITS-2) and pathogenicity data were combined to distinguish Phomopsis spp. from grapevines. Fifteen species of Phomopsis were delineated by phylogenetic analysis of ITS sequence data. Diaporthe helianthi, a sunflower pathogen, was reported from grapevines for the first time, with a further six, unknown species also distinguished. Three different clades contained isolates previously identified as D. perjuncta. Based on type studies, it appeared that the name D. viticola was available for collections from Portugal and Germany, a new species, D. australafricana, was proposed for South African and Australian isolates, formerly treated as D. perjuncta or D. viticola. An epitype specimen and culture were designated for D. perjuncta. This species was distinguished from D. viticola and D. australafricana based on morphology and DNA phylogeny. Artificial inoculations of green grapevine shoots indicated that, of the species tested, P. amygdali, a known pathogen of peaches in the USA, and P. viticola were the most virulent.
AFRIKAANSE OPSOMMING: In 'n poging om sommige patogene geassosieer met stamsiektes en syndrome, te beveg, het die navorsing in die tesis gefokus op die taksonomie en patologiese aspekte van ConiellaiPilidiella, Botryosphaeria en Phomopsis spp Voorheen is konidium pigmentasie gebruik om Pilidiella (hialien tot ligbruin konidia) van Coniella (donkerbruin konidia) te skei. Onlangs is hierdie twee genera egter as sinoniem beskou met die ouer naam, Coniella, wat voorkeur gekry het. Die belangrikste spesies in die ConiellaiPilidiella kompleks van wingerd is C. diplodiella (Speg.) Petr. & Syd., die veroorsakende organisme van witvrot van wingerd. Vorige studies het dit moeilik gevind om te onderskei tussen C. diplodiella en C. fragariae (Oudem.) B. Sutton, wat bekend is dat dit in grond voorkom en ook blaarsiektes van Fragaria en Eucalyptus veroorsaak. Beide hierdie spesies is tevore in Suid-Afrika aangemeld. Geen van die aanmeldings van C. diplodiella is egter wetenskaplik bewys nie en daarom is dit steeds 'n kwarantyn organisme. Hierdie kwarantyn status is egter bevraagteken. Op grond van DNS volgordes van die interne getranskribeerde spasieerder area ("ITS 1", "ITS2"), die 5.8S rRNS geen, die groot ribosomale subeenheid ("LSU") en die verlengingsfaktor 1-α geen ("EF-lα") van die tipe spesies van Pilidiella en Coniella, is Coniella van Pilidiella geskei, met die meerderheid van die taxa wat binne Pilidiella resorteer. Pilidiella word gekarakteriseer deur spesies met hialien tot ligbruin konidia (gem. lengte: breedte > 1.5), in teenstelling met die donkerbruin konidia van Coniella (gem. lengte: breedte ≤ 1.5). Daar is verder bewys dat Pilidiella diplodiella, voorheen C. diplodiella, veroorsakende organisme van witvrot van wingerd, die ouer naam van C. petrakii is. Hierdie swam is teenwoordig in Suid-Afrika en P. diplodiella is dus nie meer van kwarantyn belang nie. Op grond van analises van die histoon (H3) volgordes van spesies in die P. diplodiella spesies kompleks, is P. diplodiella geskei van 'n nuut beskryfde spesie, P. diplodiopsis. 'n Nuwe spesie, P. eucalyptorum, is ook voorgestel vir isolate voorheen beskou as C. fragariae, geassosieer met blaarvlek van Eucalyptus spp. Hierdie spesie het basaal van Pilidiella gegroepeer en mag moontlik nog 'n derde genus binne hierdie kompleks verteenwoordig. Pilidiella destruens is nuut as anamorf van Schizoparme destruens beskryf, wat geassosieer word met loot terugsterwing van Eucalyptus spp. in Hawaii. Die genus Botryosphaeria Ces. & De Not. is bekend as kosmopolitaans met 'n wye gasheerreeks en geografiese verspreiding. Verskeie saprofitiese spesies is aangemeld vanaf wingerd, terwyl ander ernstige patogene van hierdie gasheer is. Laasgenoemde spesies sluit in B. dothidea (Moug.: Fr.) Ces. & De Not., B. parva Pennycook & Samuels, B. obtusa (Schwein.) Shoemaker, B. stevensii Shoemaker, B. lutea A.1.L. Phillips en B. ribis Grossenb. & Duggar. Spesies aangemeld in Suid-Afrika as wingerdpatogene, is B. obtusa, B. dothidea, B. ribis en B. vitis (Schulzer) Sacco In hierdie studie is morfologiese, DNS volgorde data ("ITSl", "ITS2", 5.8S en "EF-Iα") en plantpatologiese data gebruik om II Botryosphaeria spesies, geassosieer met wingerde in Suid-Afrika en verskeie ander werelddele, te onderskei. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina en 'n Diplodia sp. is bevestig van wingerde in Suid-Afrika, terwyl Diplodia porosum, Fusicoccum viticlavatum en F. vitifusiforme as nuwe spesies beskryf is. AIhoewel isolate van B. dothidea en B. stevensii bevestig is van wingerde in Portugal, is geen van hierdie spesies en ook nie B. ribis geïsoleer nie. AIle isolate vanaf wingerd in Portugal, voorheen beskou as B. rib is, is as B. parva op grond van hul "EF-lα" volgordes geïdentifiseer. Uit kunsmatige isolasies gemaak op wingerdlote is die gevolgtrekking gemaak dat B. australis, B. parva, B. ribis en B. stevensii meer virulent is as die ander spesies wat bestudeer is. Die Diplodia sp. versamel vanaf wingerdlote is geïdentifiseer as morfologies eenders, maar filogeneties verskillend van D. sarmentorum, terwyl D. sarmentorum bevestig is as die anamorf van Otthia spiraeae, die tipe spesie van die genus Otthia (Botryosphaeriaceae). 'n Kultuur wat as 0. spiraeae geïdentifiseer is, het binne Botryosphaeria gegroepeer, en word dus as 'n moontlike sinoniem beskou. Hierdie bevindinge bevestig vroeëre voorstelle dat die generiese konsep van Botryosphaeria uitgebrei behoort te word om genera met gesepteerde askospore en Diplodia anamorwe in te sluit. Die genus Phomopsis (Sacc.) Bubak bevat verskeie spesies wat as of plantpatogenies, of saprofities, beskryf is. Tien spesies is bekend op wingerd. Slegs twee is as patogenies bevestig, naamlik P. viticola (Sacc.) Sacc., veroorsakende organisme van loot-en-blaarvlek ("streepvlek") en P. vitimegaspora Kuo & Leu (teleomorf Diaporthe kyushuensis Kajitani & Kanem.), veroorsakende organisme van geswelde arm van wingerd. In 'n vroeëre studie is bevind dat P. amygdali (Delacr.) 1.1. Tuset & M.T. Portilla, 'n bekende patogeen van Prunus sp., moontlik ook 'n patogeen van wingerd mag wees. D. perjuncta Niessl. veroorsaak egter net verbleiking van dormante lote en is dus van min belang as 'n wingerd patogeen. Gedurende die afgelope twee jaar is verskeie Phomopsis isolate van wingerde in die Wes-Kaap provinsie van Suid-Afrika verkry. Isolasies is gemaak van Phomopsis-agtige simptome, snoeiwonde en asimptomatiese kwekeryplante. Die isolate verkry uit hierdie materiaal het groot variasie ten opsigte van morfologie en kultuureienskappe getoon. Vroeëre taksonomiese verhandelings van Phomopsis het spesies-identifikasie op gasheerspesifisiteit, kultuureienskappe en morfologie gebasseer. Onlangse studies het egter getoon dat, weens wye gasheerreekse en morfologiese plastisiteit van somnuge spesies, hierdie eienskappe me meer gebruik kan word om Phomopsis spesies te identifiseer nie. Die gebruik van anamorflteleomorf verwantskappe in die identifikasie van Phomopsis spesies ook onbruikbaar omdat Diaporthe teleomorwe vir slegs ongeveer 20% van die bekende Phomopsis spesies beskryf is. Die huidige studie het dus morfologiese data, DNS volgordes ("ITS 1", 5.8S, "ITS2") en patogenisiteitsdata gekombineer ten einde Phomopsis spp. vanaf wingerd te identifiseer. Vyftien Phomopsis spesies is deur die filogenetiese analise van die interne getranskribeerde spasieerder area ("ITS") volgordes geskei. Diaporthe helianthi, 'n bekende patogeen van sonneblomme, is vir die eerste maal op wingerd aangeteken, terwyl 'n verdere ses, tans onbekende spesies van Phomopsis ook geidentifiseer is. Drie verskillende groepe het isolate bevat wat voorheen as D. perjuncta geidentifiseer is. Gebasseer op studies van tipes, het dit voorgekom dat die naam D. viticola beskikbaar is vir isolate uit Portugal en Duitsland. 'n Nuwe spesie, D. australafricana, is voorgestel vir Suid-Afrikaanse en Australiese isolate wat voorheen behandel is as D. perjuncta of D. viticola. 'n Epitipe monster en kultuur is vir D. perjuncta benoem. Hierdie spesie is van D. viticola en D. australafricana onderskei op grond van morfologie en DNS filogenie. Kunsmatige inokulasies van groen wingerdlote het getoon dat P. amygdali, bekende perske patogeen, en P. viticola die mees virulent was.

A study to assess the potentials of some commensal bacteria that belong to Staphylococcus, Acinetobacter and Stenotrophomonas genera as reservoirs of antibiotic resistance determinants in the environment of Nkonkobe Municipality of the Eastern Cape Province, South Africa, was carried out using standard microbiological and molecular techniques. A total of 120 Staphylococcus isolates which consisted of Staphylococcus haemolyticus (30%), Staphylococcus aureus (23.3%) from pig; Staphylococcus capitis (15%) from goat; Staphylococcus heamolyticus (5%) and Staphylococcus xylosus (15%) from cattle and other Staphylococci (11%) from dead chicken and pigs were isolated. About 23.3% of these isolates were coagulase positive and 76.7% were coagulase negative. This difference in prevalence along coagulase production divide was statistically significant (p < 0.05). Eighty-six Acinetobacter species (Acinetobacter baumannii/calcoaceticus and Acinetobacter haemolyticus) were also isolated from Alice and Fort Beaufort towns samples, while 125 Stenotrophomonas maltophilia isolates were from grass root rhizosphere (96%) and soil butternut root rhizosphere (4%). Between 75-100% of the Staphylococccus species were resistant to Penicillin G, tetracycline, sulphamethaxole and nalidixic acid; about 38 % were methicillin resistant, consisting of 12.6% methicillin resistant Staphylococcus aureus (MRSA) from pig and a total of 12% vancomycin resistant were observed. Also, 12% of the isolates were erythromycin resistant while 40.2 % were resistant to the third generation cephalosporin, ceftazidime. The antibiotic resistance genes vanA, VanB, eryA, eryB, eryC were not detected in all the phenotypically resistant Staphylococccus species, but mec A gene and mph genes were detected. In the Acinetobacter species, a wide range of 30-100% resistance to penicillin G, ceftriazone, nitrofurantoin, erythromycin, and augmentin was observed. Polymerase chain reaction (PCR) revealed the presence of Tet(B) and Tet(39) genes in these species, while Tet (A), Tet(M) and Tet(H) were absent. Also, 9.3% of the Acinetobacter species showed phenotypic production of extended spectrum beta lactamases (ESBLs) while 3.5% were positive for the presence of blaCTX-M-1 genes. The Stenotrophomonas maltophilia isolates showed varying resistance to meropenem (8.9%), cefuroxime (95.6 %), ampicillin-sulbactam (53.9%), ceftazidime (10.7%), cefepime (29.3 %), minocycline (2.2%), kanamycin (56.9%), ofloxacin (2.9%), levofloxacin (1.3%), moxifloxacin (2.8%), ciprofloxacin (24.3%), gatifloxacin (1.3%), polymyxin B (2.9 %), cotrimoxazole (26.1%), trimethoprim (98.6%), aztreonam(58%) and Polymyxin B (2.9 %). The isolates exhibited significant susceptibility to the fluoroquinolones (74.3-94.7 %), polymycin (97.1%) and meropenem (88.1%). Only sul3 genes were the only sulphonamide resistance gene detected among the trimethoprim-sulphamethoxazole resistant isolates. The observed multiple antibiotic resistance indeces (MARI) of >2 for Staphylococcus species, Acinetobacter species and Stenotrophomonas maltophilia suggest that they have arisen from high-risk sources where antibiotics are in constant arbitrary use resulting in high selective pressure. The presence of tetracycline resistance genes in Acinetobacter species justifies the observed phenotypic resistance to oxytetracycline and intermediate resistance to minocycline. High phenotypic resistance and the presence of some resistance genes in Staphylococcus species is a possible threat to public health and suggests animals to be important reservoirs of antibiotic resistance determinants in the environment. Indiscriminate use of antibiotics induces this kind of antibiotic resistance and should be discouraged. Personal hygiene is encouraged as it reduces the load of Acinetobacter species contacted from the environment that may be difficult to control. Commensal Stenotrophomonas maltophilia are as important as their clinical counterparts due to their roles in opportunistic infection, antibiotic resistance and their associated genes, especially sul gene. Personal hygiene is hereby advocated especially when in contact with soil, plants and plants’ rhizospheric soil

Moonlighting (multitasking, multifunctional) es la capacidad de algunas proteínas de ejecutar dos o más funciones bioquímicas. Normalmente, las proteínas moonlighting son descubiertas por "serendipia". La multifuncionalidad de las proteínas adquiere una mayor importancia a partir de los recientes descubrimientos acerca del proteoma humano (y de animales modelo como el ratón). El mecanismo de “splicing” no da lugar a un gran número de proteínas como se pensaba hasta ahora, sinó que los genes humanos mayoritariamente expresan la denominada isoforma principal, a nivel de proteína. Por ello el moonlighting puede contribuir, o ser la clave, a la complejidad de la célula. Por esta razón, sería de utilidad que la bioinformática pudiera predecir la multifuncionalidad, especialmente debido a la gran cantidad de nuevas secuencias provenientes de los proyectos genómicos. Durante el presente trabajo se han analizado y descrito diferentes aproximaciones que utilizan secuencias, estructuras, interactómica, algoritmos bioinformáticos e información contenida en las bases de datos como UniProt, OMIM, etc, para tratar de contribuir a la identificación de las proteínas multifuncionales. Un primer objetivo de este trabajo ha sido la actualización de la base de datos de proteínas moonlighting, MultitaskProtDB-II (http://wallace.uab.es/multitaskII), que ha servido como banco de trabajo para todos los análisis realizados. Por ejemplo, hemos intentado mapar los dominios funcionales de cada función dentro de la estructura de algunas proteínas importantes de nuestra base de datos. Esto ha permitido, en un cierto número de casos, identificar los dominios relacionados con una o más enfermedades humanas basadas en la proteína multifuncional analizada. De MultitaskProtDB-II, se ha encontrado que un porcentaje significativo de proteínas moonlighting (78%) están relacionadas con enfermedades humanas y que un 48% de ellas son dianas para fármacos actuales. Además, un 25% de proteínas moonlighting de la base de datos actualizada están implicadas en la virulencia de microorganismos patógenos, y hemos propuesto una explicación basada en el hecho de que las proteínas moonlighting, al ser evolutivamente muy conservadas, el huésped evitaría desarrollar una respuesta inmune que podría desencadenar una enfermedad autoinmune. Esto tiene gran importancia en la selección de proteínas candidatas a ser inmunogénicas protectivas en estudios de vacunología reversa. Desde el punto de vista de la evolución de las proteínas moonlighting y a partir del análisis de identificadores GO y de la ortología de interactomas, se sugiere que la segunda función estaría conservada filogenéticamente y que habría muchas más proteínas multifuncionales de lo que se pensaba anteriormente. Finalmente se ha analizado la posible relación evolutiva entre la multifuncionalidad y el desplazamiento de gen no ortólogo.
Moonlighting (multitasking, multifunctional) is the ability of some proteins to perform two or more biochemical functions. Normally, moonlighting proteins are discovered by "serendipity". The multifunctionality of proteins acquires greater importance from recent discoveries about the human proteome (and animal models such as mice). The splicing mechanism does not give rise to a large number of proteins as was thought until now, but human genes mainly express the so-called main isoform, at the protein level. Therefore, moonlighting can contribute, or be the key, to the complexity of the cell. For this reason, it would be useful to predict multifunctionality from a bioinformatics point of view, especially due to the large number of new sequences coming from the genomic projects. During this work we have analyzed and described different approaches that use sequences, structures, interactomics, bioinformatic algorithms and the information contained in databases such as UniProt, OMIM, etc., to try to contribute to the identification of multifunctional proteins. A first objective of this work was to update the moonlighting protein database, MultitaskProtDB-II (http://wallace.uab.es/multitaskII), which has been used as a work platform for all the analyses. For example, we mapped the functional domains of each function within the structure of the protein and this allowed, in a certain number of cases, the identification of the domains related to one or more human diseases caused by this multifunctional protein. From MultitaskProtDB-II, it has been found that a significant percentage of moonlighting proteins (78%) are related to human diseases and that 48% of them are targets for current drugs. In addition, 25% of moonlighting proteins from the updated database are involved in the virulence of pathogenic microorganisms, and we have proposed an explanation based on the fact that the moonlighting proteins, being evolutionarily very conserved, the host would avoid developing a response immune that could trigger an autoimmune disease. This is of great importance in the selection of candidate proteins in order to be immunogenic and protective in reverse vaccinology studies. From the point of view of the evolution of moonlighting proteins and from the analysis of GO identifiers and the orthology of interactomes, it has been suggested that the second function would be phylogenetically conserved and that there would be many more multifunctional proteins than previously thought. Finally, the possible evolutionary relationship between multifunctionality and non-orthologous gene displacement has been analysed.

Septoria ampelina causes a disease of grapes known as septoria leaf spot. This study was done to determined which of the fungicides currently used to control the various diseases of grapes, plus one experimental fungicide, is the most effective in controlling septoria leaf spot. Both in vitro and in vivo methods were used. In vivo studies examined the systemic and/or protectant activities of the fungicides. The systemic and protectant fungicides included Bayleton, Benlate, Elite (an experimental fungicide), Nova, Rovral and Rubigan. The protectant only fungicides included Captan, Dithane and Kocide. In vitro tests to determine the minimum inhibitory concentration (MIC) for each fungicide (e.g., the concentration of the fungicide that prevents the fungus from forming colonies on the PEA-fungicide medium), indicate that Benlate (MIC = 0.1 ppm) and Elite (MIC = 1.0 ppm) have the greatest potential'to control septoria leaf spot of grape. These are followed by Dithane, Nova and Rubigan (MIC = 2.0), which in turn are followed by Bayleton and Captan (MIC = 50.0 ppm). Kocide and Rovral did not inhibit fungal growth at concentrations up through 100 ppm. Although all the fungicides tested significantly reduced the incidence of septoria leaf spot in vivo, Benlate and Elite were the most effective fungicides (both in systemic and protectant application).
Department of Biology

A model of growth and plasmid transfer between strains of Escherichia coli and Salmonella typhimurium was developed with reference to the literature. This was the organising principle for the collection of a complete set of in vitro life history parameters of one S. typhimurium and one E. coli strain. In the course of estimating these parameters two results of note were obtained. Fits of the Lotka-Volterra competition model were obtained for data on S. typhimuiurm growing in competition with E. coli. The first noteworthy discovery was the failure of this model to account for several characteristics of growth of these strains under competition. The growth rates of plasmid-bearing and plasmid-free strains were obtained. The second main result came from examination of the results of the growth rate data, which revealed that the cost to S. typhimuiurm 576 of bearing the resistance plasmid was low (4%). The model was also used to simulate the effect of antibiotic dose on the density of the donor, recipient and transconjugant populations over time. These simulations predicted that there would be a convex relationship between antibiotic dose and transconjugant density (i.e. that the density would first rise, then fall, with increasing dose). Following from this result, laboratory experiments and in vivo experiments in chickens were directed towards obtaining information on the relationship between these two variables. This convex relationship was not demonstrated within a single experiment, although some experimental environments produced an increase in transconjugant density with dose, and others, a decrease. Few transconjugants were formed in vivo. In order to investigate the low cost of resistance and low rate of in vivo transconjugant production, cost of resistance and plasmid transfer rate of this plasmid in several strain combinations of E. coli and S. typhimuiurm was evaluated.

Mycobacterium bovis and other species of Mycobacterium tuberculosis complex (MTBC) can result to a zoonotic infection known as Bovine tuberculosis (bTB). MTBC has members that may contaminate an extensive range of hosts, including wildlife. Diverse wild species are known to cause disease in domestic livestock and are acknowledged as TB reservoirs. It has been a main study worldwide to deliberate on bTB risk factors as a result some studies focused on particular parts of risk factors such as wildlife and herd management. The objectives of this study were to design questionnaires from commercial farms and smallholding farms; isolate and identify MTBC from collected samples using culture and PCR assays recovered from Fort Hare, Middledrift and Seven star dairy farms; and assessing genotypic drug resistance through detection of mutations conferring resistance to INH and RMP associated with first line treatment for MTBC infection. Questionnaires were administered to thirty (30) smallholding farm owners in the two villages (kwaMasele and Qungqwala) and three (3) three commercial farms ( Fort Hare dairy farm, Middledrift dairy farm and Seven star dairy farm). Detection of M. tuberculosis complex was achieved by Polymerase Chain Reaction using primers for IS6110; whereas a genotypic drug resistance mutation was detected using Genotype MTBDRplus assays. Nine percent (9%) of respondents had more than 40 cows in their herd, while 60% reported between 10 and 20 cows in their herd. Relationship between farm size and vaccination for TB differed from forty one percent (41%) being the highest to the least five percent (5%). The highest number of respondents who knew about relationship between TB cases and cattle location was ninety one percent (91%). Approximately fifty one percent (51%) of respondents had knowledge about wild life access to the farms. Relationship between import of cattle and farm size ranged from nine percent (9%) to thirty five percent (35%). Cattle sickness in relation to farm size differed from forty three (43%) being the highest to the least three percent (3%); while thirty three percent (33%) of respondents had knowledge about health management. Respondents with knowledge about the occurrence of TB infections in farms were forty eight percent (48%). The frequency of DNA isolation from samples ranged from the highest forty five percent (45%) from water to the least twenty two percent (22%) from soil. Fort Hare dairy farm had the highest number of positive samples forty four percent (44%) from water samples; whereas Middledrift dairy farm had the lowest positive from water, seventeen percent (17%). Twelve (22%) out of 55 isolates showed resistance to INH and RMP that is, multi-drug resistance (MDR) and nine percent (9%) were sensitive to either INH or RMP. The mutations at rpoB gene differed from 58% being the highest to the least (23%). Fifty seven percent (57%) of samples showed a S315T1 mutation while only 14% possessed a S531L in the katG gene. The highest inhA mutations were detected in T8A (80%) eighty percent and the least was observed in A16G (17%). The results of this study reveals that risk factors for bTB in cattle and dairy farm workers is a serious issue abound in the Eastern Cape of South Africa; with the possibility of widespread dissemination of multidrug resistant determinants in MTBC from the environment.

The initial phase in the development of a biological control strategy is screening of biological control agents. Secondary to this phase is the establishment of accurate, effective application techniques. However, successful control requires a thorough understanding of all factors affecting the relationship between host plant, pathogen and other microbes. The purpose of this study was to screen and identify potential bacterial antagonists against Alternaria, a fungal citrus pathogen, attachment of the antagonists to bees, and bee dissemination of the antagonist to citrus flowers. A total of 568 bacterial epiphytes were screened on agar plates for antagonism against Alternaria. Only eight of these isolates, which were identified as Bacillus subtilis, B licheniformis, B. melcerons, B. polymyxa, B. thermoglycodasius, B. sphaericus, B. amiloliquefaciens, and B. coagulans, showed inhibitory effects on the growth of Alternaria. The most effective isolates were B. subtilis and B. licheniformis. Further screening was done with B. subtilis and B. subtilis commercial powder (Avogreen). These bacteria were sprayed on citrus flowers for colonisation studies. Mean populations of B. subtilis and the commercial powder recovered from the flowers were 104 and 103 cfu/stamen respectively. The organisms colonised the styler end and ovary of the flowers when observed under scanning electron microscope (SEM). Avogreen was placed in an inoculum dispenser, which was attached to the entrance of the hive. Honeybees emerging from the beehive acquired 104 cfu/bee. The powder attached to the thorax and thoracic appendages, as revealed by SEM. One active beehive was placed in an enclosure with fifteen flowering citrus nursery trees in pots for dissemination trials. Mean populations of commercial B. subtilis recovered from the flowers visited by bees were 104 cfu/stamen. Electron microscope studies revealed that the antagonist was colonising the styler end and ovary of the flowers. Field dissemination studies were unsuccessful due to low yields.
Dissertation (Magister Institutiones Agrariae)--University of Pretoria, 2006.
Plant Production and Soil Science
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Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia)
IPEN/D-MPLO
Instituto de Pesquisas Energeticas e Nucleares, IPEN/CNEN-SP; Faculdade de Odontologia, Universidade de Sao Paulo

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Tese (Doutorado em Tecnologia Nuclear)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

The utility of focal-plane-array Fourier transform infrared (FPA-FTIR) spectroscopy as a rapid method for the differentiation of antibiotic resistant foodborne pathogens was studied.
Optimum spectral acquisition and processing parameters as well as appropriate film thickness of bacterial films were empirically established for the discrimination between two Shigella species (S. flexneri and S sonnei) in order to optimize the scanning parameters of an FPA-FTIR spectrometer. A detailed study of the potential of FPA-FTIR spectroscopy for the discrimination between antibiotic resistant and sensitive strains from two Salmonella species (S. Typhimurium and S. Heidelberg) was subsequently undertaken. The results of these studies demonstrated that the infrared spectra recorded by an FPA-FTIR spectrometer contained sufficient information to differentiate between antibiotic resistant and sensitive strains of Salmonella. Accordingly, FPA-FTIR spectroscopy may potentially serve as a high-throughput technique for the identification of foodborne as well as antibiotic resistant bacteria.
Interpretation of the regions selected in relation to the different resistance mechanisms would require more detailed studies. However, the identification of specific biochemical markers based on such spectral interpretation is generally not feasible owing to the complexity of the FTIR spectra of microorganisms.