Thèses sur le sujet « Multiple disease resistance »

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第22128号
農博第2374号
新制||農||1073(附属図書館)
学位論文||R1||N5236(農学部図書室)
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 髙野 義孝, 教授 田中 千尋, 教授 寺内 良平
学位規則第4条第1項該当

Thesis (MSc)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies.
AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.

Colibacillosis is considered one of the more costly diseases encountered in the production of market turkeys. It is responsible for a significant amount of mortality in birds between the ages of 6-12 weeks. Research conducted over the past 5 years has shown that within the Shenandoah Valley production area, multiple primary infectious agents are responsible for the predisposition of turkeys to colibacillosis. These agents were first identified as potential contributors through field case studies. They include hemorrhagic enteritis (HE) virus, Newcastle disease virus, and Bordetella avium. Further retrospective serologic studies affirmed the role of these primary agents and uncovered the potential involvement of Mycoplasma meleagridis. Trials were conducted to determine the reproducibility of some multiple agent interactions under laboratory conditions. It was found that Newcastle disease virus or B. avium infection followed by HE virus and Escherichia coli challenge produced clinical colibacillosis. It is believed that hemorrhagic enteritis virus is the pivotal agent in this process of predisposition. Almost all turkeys are vaccinated for hemorrhagic enteritis in the field. The virulent strains of the virus are known to be immunosuppressive. It is suspected that the vaccine strains are mildly so. Infection with HE vaccine virus was shown to cause an increase in CT8+ cells in peripheral blood. These cells are believed to be suppressor T-cells and may account for the reputed immunosuppressive effects of the virus. Thus, interactions of multiple infectious agents including Newcastle disease virus, B. avium, M. meleagridis, and HE virus appear to be involved in the predisposition of turkeys to secondary E. coli infections.
Ph. D.

Ce travail porte sur des approches translationnelles dans les synucléinopathies notamment l’atrophie multisystématisée (AMS). Au-delà de leur rôle dans la régulation du glucose, l’insulin et l’insulin like growth factor-1 (IGF-1) ont des propriétés neurotrophiques. Des études ont montrées que la signalisation de l’insuline/IGF-1 est altérée dans la maladie d'Alzheimer et des données suggèrent l’altération de l’insuline/IGF-1 dans la maladie de Parkinson (MP) et l’AMS. Nous avons mis en évidence une résistance à l’insuline dans les neurones des patients MP et AMS ainsi que dans les oligodendrocytes chez les patients AMS.Mon travail a également consisté à cibler la troncation de l’α-synuclein (α-syn) comme cible thérapeutique. Nous avons démontré dans un modèle murin d’AMS que la diminution de l’α-syn tronquée permettait de réduire l’agrégation d’α-syn et la dégénérescence des neurones dopaminergiques.Enfin, nous avons étudié l’implication dans l’AMS des métalloprotéinases matricielles (MMP), des enzymes impliquées dans remodelage de la matrice, la démyélinisation, la troncation de l’α-syn et la perméabilité de la barrière hémato-encéphalique. Ce travail nous a permis de montrer une augmentation de l’expression et de l’activité de MMPs chez les patients AMS. Nous avons également montré que les cellules gliales sont la source de cette augmentation et que la MMP-2 est retrouvée dans les agrégats des patients AMS.Nous montrons ici de caractéristiques distinctes de l’AMS comme des altérations qui se produisent dans les oligodendrocytes. Nous présentons aussi VX-765 comme un candidat prometteur pour ralentir la progression de la pathologie dans un contexte de synucléinopathie
This work focused on translational approaches in synucleinopathies and more specifically in multiple system atrophy (MSA). Beyond their role in glucose homeostasis, insulin/IGF-1 are neurotrophic factors in the brain. Studies have shown altered insulin/IGF-1 signalling in Alzheimer’s disease and data suggest impaired insulin signaling/IGF-1 in Parkinson's disease (PD) and MSA. The aim of my work was to characterize insulin/IGF-1 signalling in MSA and PD brain tissue. Both groups showed neuronal insulin resistance. Oligodendrocytes in MSA patients were also insulin resistant.In line with the translational approach, we also targeted α-synuclein (α-syn) truncation pharmacologically in MSA transgenic mice, which led to reduced α-syn aggregation and the protection of dopaminergic neurons.We also assessed the activity and distribution of matrix metalloproteinases (MMPs) in the brain of MSA patients compared to healthy controls. MMPs are involved in the remodelling of the extracellular matrix, demyelination, α-syn truncation and blood brain barrier permeability. We showed altered expression and activity of MMPs in two distinct structures in MSA brains. We were also able to show that glial cells were the source of increased MMPs and show a unique expression of MMPs in α-syn aggregates of MSA patients compared to PD, evidence that might hint at a mechanism that is differently altered between PD and MSA.We here show distinct pathological features of MSA such as key alterations occurring in oligodendrocytes, further supporting MSA as a primary oligodendrogliopathy. We also present VX-765 as a candidate drug for disease modification in synucleinopathies

Four F2:4 populations of peanut (Arachis hypogaea L.) resulting from the complex cross Tamrun 96 X Tx901639-3 X Sun Oleic 95R were grown in three disease nurseries over a 2 year period. Three separate selection techniques were applied to determine which technique would provide the most effective method for selecting a multiple disease resistant, runner-type peanut. Technique I involved selection at a tomato spotted wilt virus nursery during the first cycle of selection and transferring the selections to a Sclerotinia minor (Jagger) nursery for a second cycle of selection in year two. Technique II was the reciprocal of Technique I. Technique III involved selection of the populations at a multiple disease nursery for two consecutive years. Selections were based on disease ratings, growth habits, pod and seed characteristics, and oleic/linoleic acid ratios. Disease ratings were scored as percentage infection on a scale of 0 (0% plot infected) to 10 (100% plot infected). Disease severity was also rated on a scale of 1 (symptoms noted, but no yield effects) to 10 (plant death, no yield). There were two final selections for each population using each selection technique that were yield tested over a 2 year period to determine which technique was superior. The yield tests were conducted using completely randomized block design at all three disease nurseries with an additional disease-free site included. Data for disease ratings, yield, grade, and value per hectare were combined within locations across years. All three selection techniques provided lines with more disease resistance than the parents; however, there was no difference detected between the effectiveness of the three techniques in terms of disease resistance, yield, grade, or value per hectare.

La presente tesi riguarda tre approcci complementari per un controllo più sostenibile del patogeno Plasmopara viticola: cisgenesi, RNAi e priming di difesa delle piante. Nel primo capitolo viene presentata una breve introduzione generale, toccando i principali aspetti relativi alla viticoltura in Europa, alle caratteristiche della malattia, alle nuove strategie biotecnologiche e al priming nella difesa delle piante. Nel secondo capitolo viene presentata una review che descrive in dettaglio i più recenti approcci biotecnologici per la protezione delle colture, tra cui la cisgenesi, l'editing del genoma, l'RNAi e l'epigenetica. Nel terzo capitolo sono riportate le attività relative alla cisgenesi per l’introduzione della resistenza alla peronospora nella vite, lo studio si è concentrato inizialmente sull'induzione dell'embriogenesi somatica in germoplasma d'élite, ottimizzando la coltivazione dei tessuti floreali per la generazione di calli embriogenici. I geni di resistenza TNL2a e TNL2b appartenenti al locus RPV3-1, che conferiscono resistenza a Plasmopara viticola, sono stati quindi selezionati per lo sviluppo di varietà cisgeniche, con la costruzione di un vettore cisgenico che ospita questi due geni. Viene quindi descritta la trasformazione dei calli embriogenici con i ceppi ingegnerizzati di Agrobacterium tumefaciens e le future attività per la rigenerazione di piante cisgeniche trasformate. Nel quarto e nel quinto capitolo vengono presentati due articoli che affrontano diversi aspetti legati allo sfruttamento del sistema immunitario delle piante: il primo studio mira a chiarire gli effetti del priming indotto da micorrize sul bilancio tra crescita e difesa nella vite mentre il secondo studio si concentra sull'utilizzo di protocolli di protezione alternativi per il controllo della peronospora in un vigneto commerciale. In particolare, nel quarto capitolo “Mycorrhizal symbiosis balances rootstock-mediated growth-defence tradeoffs”, sono stati valutati i potenziali benefici di un inoculo formato da due specie di micorrize arbuscolari, con o senza aggiunta di monosaccaridi, su giovani barbatelle innestate sui portainnesti 1103P e SO4. L'influenza dei diversi trattamenti è stata valutata combinando l'analisi delle caratteristiche agronomiche con tecniche biochimiche e molecolari. I risultati hanno mostrato che, nonostante il comportamento opposto dei due portainnesti selezionati, nei campioni trattati con le micorrize l'intero microbioma della radice è attivamente coinvolto nel bilanciamento dei costi/benefici tra crescita e difesa. Infine, nel quinto capitolo, viene presentato l’articolo "Novel Sustainable Strategy to control Plasmopara viticola in grapevine, unveil new insights on priming responses and artropods ecology". Lo studio affronta la riduzione del consumo di fungicidi in viticoltura e dei rischi associati attraverso lo sfruttamento di protocolli alternativi per il controllo della peronospora nella vite confrontandoli con un protocollo di protezione standard adottato da una cantina commerciale. Nel primo protocollo sono stati utilizzati solo induttori di resistenza, mentre il secondo e il terzo protocollo hanno seguito il protocollo standard ma sostituendo i fosfonati con anidride fosforica ed estratto di Ecklonia maxima. I risultati hanno mostrato che all'invaiatura l'incidenza e la gravità della peronospora in tutti i protocolli testati erano significativamente ridotte rispetto ai controlli non trattati sia sulla chioma che sui grappoli. Lo studio ha anche mostrato degli spunti interessanti sulla rimodulazione dell'acido salicilico e dell'acido jasmonico nei due protocolli per la sostituzione dei fosfiti. È interessante notare come gli induttori di resistenza attivando le difese della pianta abbiano indotto anche un breve ritardo nella maturazione dei grappoli, agendo, sul metabolismo dei carboidrati, sulla regolazione dei geni di difesa, sulla risposta sistemica acquisita e sulla disintossicazione dalle specie reattive dell’ossigeno. Nella conclusione sono quindi riassunti i principali risultati di ciascun capitolo, esaminandone gli aspetti più critici, inclusa una breve discussione delle attività preliminari che sono state condotte sull’uso dell’RNAi per il silenziamento di due geni essenziali di Plasmopara viticola.
The present thesis relates on three complementary approaches for a more sustainable control of Plasmopara viticola: cisgenesis, RNAi and plant defence priming. A brief general introduction is presented in the first chapter, touching the main aspects relative to viticulture in Europe, characteristics of the disease, new biotechnological strategies and priming of plant defence. The second chapter consists of a review article describing with detail the most recent biotechnological approaches for crop protection, including cisgenesis, genome editing, RNAi and epigenetics. In the third chapter the activities concerning cisgenesis for grapevine downy mildew resistance are reported, the study initially focuses on the induction of somatic embryogenesis from elite germplasm, optimising the cultivation of floral tissues for the generation of embryogenic calli. The resistance genes TNL2a and TNL2b belonging to the RPV3-1 locus, which confers resistance to Plasmopara viticola, were then selected for the development of cisgenic varieties, with the construction of a cisgenic vector harbouring those two genes. Finally, the chapter reports on the Agrobacterium tumefaciens transformation of embryogenic calli that are currently cultivated on selective medium, and on the future activities for the regeneration of transformed cisgenic plants. In the fourth and fifth chapters, two papers addressing different aspects related to the exploitation of plant immune system are presented: the first study aimed at clarifying the effects of arbuscular mycorrhiza priming on the grapevine growth-defence trade-off while the second study was focused on the use of alternative protection protocols for the control of downy mildew in a commercial vineyard. Particularly, in the fourth chapter “Mycorrhizal symbiosis balances rootstock-mediated growth-defence tradeoffs”, the potential benefits of an inoculum formed by two arbuscular mycorrhiza fungal species, with or without a monosaccharide addition, were evaluated on young grapevine cuttings grafted onto 1103P and SO4 rootstocks. The influence of the different treatments was assessed by combining the analysis of agronomic features with biochemical and molecular techniques. The results showed that despite the opposite behaviour of the two selected rootstocks, in mycorrhized samples the whole root microbiome is actively involved in the growth-defence trade off balance. Finally in the fifth chapter the submitted paper “Novel sustainable strategies to control Plasmopara viticola in grapevine unveil new insights on priming responses and arthropods ecology” is presented. The study addresses the reduction of fungicide consumption in viticulture and its associated risks by the exploitation of alternative protocols for the control of downy mildew infection in grapevine, compared to a standard winery protection protocol. In the first protocol, only resistance inducers were used, while the second and third protocols followed the standard protocol but substituting phosphonates with phosphorus pentoxide and Ecklonia maxima extract. The results showed that, at véraison, downy mildew incidence and severity were significantly reduced on both canopy and bunches in the plants treated with all tested protocols compared to non-treated controls. The study also revealed interesting insights about the direct effect of protocols for phosphite substitution on the crosstalk between salicylic and jasmonic acid signalling pathways. Interestingly, by priming plant defences, the resistance inducers caused a short delay in bunch ripening, involving changes in carbohydrate metabolism, regulation of defence related genes, systemic acquired resistance and reactive oxygen species detoxification. In the thesis conclusion, the main findings are then summarised for each chapter, by examining the most critical aspects and including a brief discussion on the preliminary activities that were conducted to exploit the RNAi technique for silencing two essential genes of Plasmopara viticola.

Over a decade, proteasome inhibitors (PIs), bortezomib, carfilzomib (Cfz) and ixazomib, have contributed to a significant improvement in the overall survival for multiple myeloma (MM) patients. However, the response rate of PI was fairly low, leaving a huge gap in MM patient care. Given this, mechanistic understanding of PI resistance is crucial towards developing new therapeutic strategies for refractory/relapsed MM patients. In this dissertation work, we found H727 human bronchial carcinoid cells are inherently resistant to Cfz, yet susceptible to other PIs and inhibitors targeting upstream components of the ubiquitin-proteasome system (UPS). It indicated H727 cells may serve as a cell line model for de novo Cfz resistance and remains UPS dependent for survival. To examine the potential link between proteasome catalytic subunit composition and cellular response to Cfz, we altered the composition of proteasome catalytic subunits via interferon-γ treatment or siRNA knockdown in H727 cells. Our results showed alteration in composition of proteasome catalytic subunits results in sensitization of H727 cells to Cfz. It supported that proteasome inhibition by alternative PIs may still be a valid therapeutic strategy for patients with relapsed MM after having received treatment with Cfz. With this in mind, we designed and synthesized a small library of epoxyketone-based PIs by structural modifications at the P1′ site. We observed that a Cfz analog, harboring a hydroxyl substituent at its P1′ position was cytotoxic against cancer cell lines with de novo or acquired resistance to Cfz. These results suggested that peptide epoxyketones incorporating P1′-targeting moieties may have the potential to overcome Cfz resistance mechanisms in cells. The immunoproteasome (IP), an inducible proteasome variant which is harboring distinct catalytic subunits, LMP2, MECL1 and LMP7 of the proteasome typically expressed in cells of hematopoietic origin, plays a role in immune response and is closely linked to inflammatory diseases. It has been reported that the IP is upregulated in reactive glial cells surrounding amyloid β (Aβ) deposits in brains of Alzheimer’s disease (AD) patients and AD animal models. To investigate whether the IP is involved in the pathogenesis of AD, we examined the impact of IP inhibition on cognitive function in AD mouse models. We observed that YU102, an epoxyketone peptide targeting the IP catalytic subunit LMP2, improved cognitive dysfunction in AD mice without clearance of Aβ deposition or tau aggregation. Our cell line model study also showed a potential mode of action of YU102 which is suppressing pro-inflammatory cytokine production in microglial cells. It suggested that LMP2 contributes to microglia-mediated inflammatory response. These findings supported that LMP2 may offers a valuable therapeutic target for treatment of Alzheimer’s disease, expanding the therapeutic potential of the LMP2-targeting strategy.

The main objectives of this study were to examine the nature of the basic-applied research continuum, to evaluate projected costs and benefits of the research continuum for a specific example, namely, the development of multiple disease resistant potato cultivars, and to examine the changing roles and interactions between public research institutions and private industry. Given the existing budgetary constraints and the increasing demands for accountability that research administrators and policy makers face, it seems necessary for decision makers to give adequate consideration to the existing interdependency of basic and applied research in determining the most appropriate levels of research to fund. The establishment of of an adequate balance of both basic and applied research is important in any attempts to maximize the returns from the research continuum while at the same time developing and maintaining new biotechnologies. The projected rate of return for potato disease resistant research was calculated at 34 percent which falls within the range given by similar studies. With the advent of the new biotechnologies such as genetic engineering, and the increased competition for the limited research dollars, there has been an evolving new relationship between universities and private industry in which universities are seeking more private funding and industry demanding more control of technologies developed through their funding. Separate but interdependent roles of both private companies and universities seems necessary for the achievement of desirable and a adequate maintenance of the research continuum.
M.S.

Examination of variation at polymorphic microsatellite loci is a widely accepted method for determining parentage and examining genetic diversity within rainbow trout (Oncorhynchus mykiss) breeding programs. Genotyping costs are considerable; therefore, we developed a single-step method of co-amplifying twelve microsatellite loci in two hexaplex reactions. The protocol is explicitly described to ensure reproducible results. I applied the protocol to samples previously analyzed at the National Center for Cool and Coldwater Aquaculture (NCCCWA) with previously reported marker sets for a comparison of results. Each marker within the multiplex system was evaluated for duplication, null alleles, physical linkage, and probability of genotyping errors. Data from four of the 12 markers were excluded from parental analysis based on these criteria. Parental assignments were compared to those of a previous study that used five independently amplified microsatellites. Percentages of progeny assigned to parents were higher using the subset of eight markers from the multiplex system than with five markers used in the previous study (98% vs. 92%). Through multiplexing, use of additional markers improved parental allocation while also improving efficiency by reducing the number of PCR reactions and genotyping runs required. I evaluated the methods further through estimation of F-statistics, pairwise genetic distances, and cluster analysis among brood-years at the NCCCWA facility. These estimates were compared to those from nine independently amplified microsatellites used in a previous study. Fst metrics calculated between brood-years showed similar values of genetic differentiation using both marker sets. Estimates of individual pairwise genetic distances were used for constructing neighbor-joining trees. Both marker-sets yielded trees that showed similar subpopulation structuring and agreed with results from a model-based cluster analysis and available pedigree information. These approaches for detecting population substructure and admixture portions within individuals are particularly useful for new breeding programs where the founders' relatedness is unknown. The 2005 NCCCWA brood-year (75 full-sib families) was challenged with Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD). The overall mortality rate was 70%, with large variation among families. Resistance to the disease was assessed by monitoring post-challenge days-to-death. Phenotypic variation and additive genetic variation were estimated using mixed models of survival analysis. The microsatellite markers used were previously isolated from BAC clones that harbor genes of interest and mapped onto the rainbow trout genetic linkage map. A general relationship between UBA gene sequence types and MH-IA-linked microsatellite alleles indicated that microsatellites mapped near or within specific major histocompatibility (MH) loci reliably mark sequence variation at MH genes. The parents and grandparents of the 2005 brood-year families were genotyped with markers linked to the four MH genomic regions (MH-IA, MH-IB, TAP1, and MH-II) to assess linkage disequilibrium (LD) between those genomic regions and resistance to BCWD. Family analysis suggested that MH-IB and MH-II markers are linked to BCWD survivability. Tests for disease association at the population level substantiated the involvement of MH-IB with disease resistance. The impact of MH sequence variation on selective breeding for disease resistance is discussed in the context of aquaculture production.
Master of Science

Background Coagulase-negative staphylococci (CoNS) and in particular Staphylococcus epidermidis have emerged as major pathogens primarily causing nosocomial infections in patients with indwelling medical devices. These infections are often caused by multidrug-resistant strains of S. epidermidis (MDRSE). Other clinical entities due to CoNS are lower urinary tract infections (UTI) in women and native valve endocarditis. The purpose of this work was to investigate the frequency of antibiotic resistance and the molecular epidemiology of both hospital and community-associated isolates of S. epidermidis in order to examine if certain clones are related to MDRSE infections. Furthermore, we aimed to explore if specific clones of S. saprophyticus are associated with UTI in women. Methods A total of 359 hospital-associated methicillin-resistant isolates of CoNS obtained from 11 hospitals in northern Europe and 223 community-associated staphylococcal isolates were examined. Furthermore, 126 isolates of S. saprophyticus isolated from women with uncomplicated UTI from five different locations in northern Europe were analyzed. Pulsed-field gel electrophoresis (PFGE) was used for genotyping. Additionally, some of the S. epidermidis isolates were analyzed with multilocus sequence typing (MLST). Antibiotic susceptibility was determined for all isolates by the disc diffusion test. Results 293 of the 359 (82%) hospital-associated and 124 of the 223 (56%) community-associated isolates belonged to the species S. epidermidis. Among the hospital-associated S. epidermidis isolates, two dominating PFGE types (type A and B) were distinguished, comprising 78 (27%) and 51 (17%) isolates, respectively. Type A, which was detected in a Norwegian and eight Swedish hospitals, corresponded with a novel sequence type (ST215). Type B was discovered in a German, a Danish and seven Swedish hospitals and corresponded with ST2. In contrast, community-associated isolates of S. epidermidis were genetically extremely diverse with no predominating genotype, and showed a low rate of antibiotic resistance; only two (1.6%) methicillin-resistant strains were detected. Among 126 analyzed isolates of S. saprophyticus, 47 different PFGE profiles were identified. Several clusters of genetically highly related isolates were detected among isolates obtained from different locations and periods of time. Conclusion We have demonstrated the occurrence, persistence and potential dissemination of two multidrug-resistant S. epidermidis (MDRSE) genotypes, including a novel sequence type (ST215), within hospitals in northern Europe. Community-associated isolates of S. epidermidis showed a low rate of methicillin-resistance and were genetically heterogeneous. These results indicate that MDRSE by large are confined to the hospital setting in our region. Moreover, although the S. saprophyticus population was quite heterogeneous, indistinguishable isolates of S. saprophyticus causing lower UTI in women were identified in different countries 11 years apart, indicating the persistence and geographical spread of some clones of S. saprophyticus.

The primary objective of this dissertation is to utilise, adapt and extend current stochastic models and statistical inference techniques to describe the transmission of nosocomial pathogens, i.e. hospital-acquired pathogens, and multiply-resistant organisms within the hospital setting. The emergence of higher levels of antibiotic resistance is threatening the long term viability of current treatment options and placing greater emphasis on the use of infection control procedures. The relative importance and value of various infection control practices is often debated and there is a lack of quantitative evidence concerning their effectiveness. The methods developed in this dissertation are applied to data of methicillin-resistant Staphylococcus aureus occurrence in intensive care units to quantify the effectiveness of infection control procedures. Analysis of infectious disease or carriage data is complicated by dependencies within the data and partial observation of the transmission process. Dependencies within the data are inherent because the risk of colonisation depends on the number of other colonised individuals. The colonisation times, chain and duration are often not visible to the human eye making only partial observation of the transmission process possible. Within a hospital setting, routine surveillance monitoring permits knowledge of interval-censored colonisation times. However, consideration needs to be given to the possibility of false negative outcomes when relying on observations from routine surveillance monitoring. SI (Susceptible, Infected) models are commonly used to describe community epidemic processes and allow for any inherent dependencies. Statistical inference techniques, such as the expectation-maximisation (EM) algorithm and Markov chain Monte Carlo (MCMC) can be used to estimate the model parameters when only partial observation of the epidemic process is possible. These methods appear well suited for the analysis of hospital infectious disease data but need to be adapted for short patient stays through migration. This thesis focuses on the use of Bayesian statistics to explore the posterior distributions of the unknown parameters. MCMC techniques are introduced to overcome analytical intractability caused by partial observation of the epidemic process. Statistical issues such as model adequacy and MCMC convergence assessment are discussed throughout the thesis. The new methodology allows the quantification of the relative importance of different transmission routes and the benefits of hospital practices, in terms of changed transmission rates. Evidence-based decisions can therefore be made on the impact of infection control procedures which is otherwise difficult on the basis of clinical studies alone. The methods are applied to data describing the occurrence of methicillin-resistant Staphylococcus aureus within intensive care units in hospitals in Brisbane and London

The primary objective of this dissertation is to utilise, adapt and extend current stochastic models and statistical inference techniques to describe the transmission of nosocomial pathogens, i.e. hospital-acquired pathogens, and multiply-resistant organisms within the hospital setting. The emergence of higher levels of antibiotic resistance is threatening the long term viability of current treatment options and placing greater emphasis on the use of infection control procedures. The relative importance and value of various infection control practices is often debated and there is a lack of quantitative evidence concerning their effectiveness. The methods developed in this dissertation are applied to data of methicillin-resistant Staphylococcus aureus occurrence in intensive care units to quantify the effectiveness of infection control procedures. Analysis of infectious disease or carriage data is complicated by dependencies within the data and partial observation of the transmission process. Dependencies within the data are inherent because the risk of colonisation depends on the number of other colonised individuals. The colonisation times, chain and duration are often not visible to the human eye making only partial observation of the transmission process possible. Within a hospital setting, routine surveillance monitoring permits knowledge of interval-censored colonisation times. However, consideration needs to be given to the possibility of false negative outcomes when relying on observations from routine surveillance monitoring. SI (Susceptible, Infected) models are commonly used to describe community epidemic processes and allow for any inherent dependencies. Statistical inference techniques, such as the expectation-maximisation (EM) algorithm and Markov chain Monte Carlo (MCMC) can be used to estimate the model parameters when only partial observation of the epidemic process is possible. These methods appear well suited for the analysis of hospital infectious disease data but need to be adapted for short patient stays through migration. This thesis focuses on the use of Bayesian statistics to explore the posterior distributions of the unknown parameters. MCMC techniques are introduced to overcome analytical intractability caused by partial observation of the epidemic process. Statistical issues such as model adequacy and MCMC convergence assessment are discussed throughout the thesis. The new methodology allows the quantification of the relative importance of different transmission routes and the benefits of hospital practices, in terms of changed transmission rates. Evidence-based decisions can therefore be made on the impact of infection control procedures which is otherwise difficult on the basis of clinical studies alone. The methods are applied to data describing the occurrence of methicillin-resistant Staphylococcus aureus within intensive care units in hospitals in Brisbane and London

Availability of multiple disease resistant genotypes is especially important in international breeding centers where hundreds of crosses are prepared annually, and the availability of parents having resistance for multiple diseases would potentially facilitate breeders task in combining multiple disease resistance in a single cross. The principal aim of this study was to identify novel sources of resistance to multiple wheat fungal pathogens including wheat head and leaf blight diseases. For this goal, wheat genotypes of different geographic origins were tested for different FHB resistance types and four leaf spotting diseases including Tan spot (TS), Stagonospora nodorum blotch (SNB), Septoria tritici blotch (STB), in addition to Spot blotch (SB), independently.

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched.
Health Studies
M.Sc. (Life Sciences (Microbiology))