Embargoed till 18 November 2011 / No abstract available.

Embargoed till 18 November 2011 / No abstract available.

Notre travail est une contribution à l'étude des leishmanioses en Iran. Il s'est focalisé sur trois volets. Le principal concerne les Phlébotomes. Les deux autres sont constitués par les Rongeurs réservoirs de L. major et l'Homme.L'étude des phlébotomes s'est réalisée selon deux axes : systématique et épidémiologique. Les approches de systématique nous a conduit à réaliser des inventaires faunistiques dans plusieurs régions d'Iran (nord-ouest, Nord-est, est et centre du pays). Ces inventaires nous ont permis de réactualiser la distribution de certains groupes, notamment les phlébotomes du sous-genre Adlerius et de mentionner deux espèces nouvelles pour la faune du l'Iran : Phlebotomus turanicus et P. salangensis. A côté de ces travaix de faunistique, nous avons réalisé deux travaux de systématique évolutive qui couplent des approches morphologique et morphométrique à des approches moléculaires associant un marqueur ribosomique à un marqueur mitochondrial. Nous avons ainsi étudié P. perfiliewi s.l. et le sous-genre Adlerius sur un échantillonnage débordant largement le cadre de l'Iran. Les travaux d'épidémiologie nous ont permis de vérifier que les foyers que nous avons étudiés fonctionnent sur un mode classique dans lequel P. papatasi, et à un degré probablement moindre les femelles du groupe Caucasicus transmettent Leishmania major alors que P. sergenti assure la transmission de L. tropica. L'identification des parasites est réalisée par voie moléculaire : PCR et RFLP et/ou séquençage de marqueurs ribosomiques. L'étude des Rongeurs réservoirs de L. major nous a permis de constater le rôle essentiel de réservoir de Rhombomys opimus et de Meriones libycus dans le pays avec l'observation de prévalences très élevées, supérieures à 30 % chez ces espèces.Enfin, une étude a été conduite dans la region de Fars où sévissent classiquement des leishmanioses à L. major, L. tropica et à L. infantum. Chez 44 patients présentant des lésions cutanées, nous avons isolé et identifié essentiellement L. major et quelques souches de L. tropica. / This work focused on the leishmaniases in Iran. It includes three topics: i) the Phlebotomine sand flies, ii) the rodent reservoirs of Leishmania major and iii) the Humans.The entomological studies concern both systematics and epidemiology.The systematic part of the study of Phlebotomine sand flies includes several inventories carrired out in several provinces of Iran (North-West, North-East, East and Center of the country). Our results update the distribution of the subgenus Adlerius. We also recorded two new species for the fauna of Iran: Phlebotomus turanicus and P. salangensis. We also carried out evolutive and comparative systematics including specimens from Iran, neighbouring and Mediterranean countries. We coupled morphology to morphometrics and molecular systematics. In the latter approach, we coupled a ribosomal DNA marker to a mitochondrial one. We studied P. perfiliewi s.l. and the subgenus Adlerius. Our epidemiological works focusing on the epidemiology in several parts of the country showed that the studied foci are classical: P. papatasi, and also the females of the Caucasicus group transmit Leishmania major whereas P. sergenti is the vector of L. tropica.The identification of the Leishmania has been done using PCR then RFLP and/or sequencing of rDNA Internal Transcribed Spacer 1.We studied the rodent reservoirs of L. major. Rhombomys opimus and Meriones libycus play an important role and the percentage of L. major infection is high (> 30%).Lastly, we carried out a study on patients from the province of Fars. In this area, leishmaniases are due to L. major, L. tropica and L. infantum. We have typed strains isolated from 42 ot 44 patients. The majority of the strains have been identified as L. major and a few L. tropica.

Made available in DSpace on 2015-04-20T12:31:08Z (GMT). No. of bitstreams: 1 eleilza.pdf: 788700 bytes, checksum: 7900cf61a2a7a4d6a517f73e27656735 (MD5) Previous issue date: 2006-04-03 / Larvicidal activity of B. sphaericus from several places in Amazonia, was estimated to Anopheles darlingi and Culex quinquefasciatus. Pobit analysis was used to determine the median lethal concentration (LC50) and potency relative to the 2362 standard strain. Findings pointed out strains IB15 (LC50 = 0.040 ppm), IB19 and S1116 (LC50 = 0.048 ppm), IB16 (LC50 = 0.052 ppm) and S265 (LC50 = 0.057 ppm) as the most effective ones, being IB15 with a potency of close to 50%, higher to strain 2362 in the assays carried out with A. darlingi. IB16 and S1116 were more potent in the experiments with C. quinquefasciatus, showing to be nearly 300-400% higher. Through molecular characterisation were diagnosed the binary toxin gene in all twenty studied stains and MTX1 toxin was found only on fourteen of them. Twenty-three polymorphic sites on the sequences were observed on the basis of the BinA sequences, being only four of them informative for the parsimony. As a whole, ten haplotypes were obtained amongst the Amazonian strains, being haplotype 1 (nine strains) similar to that of 2362 (Type 2) and the rest presenting distinct sequences. The 16 polymorphic sites observed on the aminoacid sequences resulted into 19 variant aminoacids and, on the basis of the genetic distance among the strains, it was not possible to establish a correlation between variations on BinA toxin sequences and toxicity level, as well as precedence of the strains. As B. sphaericus raises biotechnological interest in the production of biolarvicides, IB15 strain microbial growth in NYSM medium during 24h of fermentation, was studied. It was found that IB15 presented a growth profile similar to 2362 strain to prduction of cells, spores, biomass and larvicidal activity throughout fermentation, with no statistically significant differences. At the end, 1.61x109 spores.mL-1 was obtained with IB15 and 6.46x108 spores.mL-1 with 2362. LT50 values were similar, being in average 2.5h in the experiments with one culture of the bacillus at the end of 24 hours. Strain IB15 showed to be suitable for growth in NYSM medium, presenting desirable levels of production of spores and toxins throughout fermentation. Further studies are needed on the large-scale production with that strain for the development of biolarvicides. Therefore, with the findings obtained in this study, one verifies the great importance of mosquito biological control with the use of entomopathogenic bacteria, showing to be a viable complement or alternative on the control of vector mosquitoes in Amazonia. / A atividade larvicida de B. sphaericus, procedentes de diversas localidades da Amazônia, foi estimada por meio de bioensaios com Anopheles darlingi e Culex quinquefasciatus. Pela análise de Probit foi determinada a concentração letal mediana (CL50) e a potência em relação à estirpe padrão 2362. Os resultados apontaram as estirpes IB15 (CL50 = 0,040 ppm), IB19 e S1116 (CL50 = 0,048 ppm), IB16 (CL50 = 0,052 ppm) e S265 (CL50 = 0,057 ppm) como mais efetivas, sendo IB15 com potência cerca de 50% superior à estirpe 2362 nos bioensaios realizados contra A. darlingi e IB16 e S1116 mais potentes nos testes com C. quinquefasciatus, mostrando-se cerca de 300-400% superiores. Na caracterização molecular o gene da toxina binária foi diagnosticado em todas as estirpes estudadas e a toxina MTX1 foi observada em apenas quatorze estirpes. Com base nas sequências de BinA, foram observados 23 sítios polimórficos nas sequências, sendo apenas quatro informativos para parcimônia. Ao total obteve-se dez haplótipos entre as estirpes da Amazônia, sendo o haplótipo 1 (nove estirpes) similar a 2362 (Tipo 2) e o restante com sequências distintas. Os 16 sítios polimórficos observados nas sequências de aminoácidos resultaram em 19 aminoácidos variantes e, com base na distância genética, não foi possível estabelecer uma correlação entre variações nas sequências da toxina BinA e o nível de toxicidade, bem como a procedência das estirpes. Como B. sphaericus desperta um interesse biotecnológico na produção de biolarvicidas para controle de mosquitos vetores, foi estudado o crescimento microbiano da estirpe IB15 em meio NYSM, durante 24 horas de fermentação. IB15 apresentou um perfil de crescimento semelhante à estirpe 2362 quanto às produções de células, esporos, biomassa e em atividade larvicida ao longo da fermentação. Ao final, obteve-se 1,61x109 esporos.mL-1 com IB15 e 6,46x108 esporos.mL-1 com 2362. Os valores de TL50 foram semelhantes, sendo em média 2,5h nos testes com uma cultura do bacilo ao final de 24 horas. A estirpe IB15 demonstrou ser adequada para crescimento em meio NYSM, apresentando níveis desejados de produção de esporos e toxinas ao longo da fermentação. São necessários estudos complementares sobre produção em grande escala com essa estirpe para o desenvolvimento de biolarvicidas. Portanto, com os resultados obtidos nesse trabalho, verifica-se a grande importância do controle biológico de mosquitos com o emprego de bactérias entomopatogênicas, mostrando ser uma alternativa ou complemento viável no controle de mosquitos vetores na Amazônia.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination. / AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies. Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde. Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie materiaal word meestal geënt op onderstokmateriaal waarvan die virus status onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n noodsaaklike beheermeganisme vir virus-infeksie. Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut konstruk bevat. ‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3 infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat v getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het. Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante wat teenwoordig is in Suid-Afrika nie. Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus weerstandbiedendheids studies. Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.

Salmonella est une bactérie zoonotique qui représente un problème majeur de santé publique. Le porc est identifié comme l'un des principaux réservoirs de Salmonella. L'objectif de ces travaux de recherche était d'identifier les mesures prioritaires à mettre en place afin de limiter la présence de Salmonella dans la filière porcine réunionnaise. Près de 80% des élevages de porcs investigués étaient infectés par Salmonella en fin d'engraissement. Cette infection asymptomatique est associée à plusieurs facteurs de risque : les conditions géo-climatiques (altitude et pluviométrie), la production de volailles sur le site, la taille de l'élevage et la présence de salmonelles dans les salles avant l'entrée du lot. Le suivi longitudinal a d'ailleurs mis en évidence que les salmonelles résiduelles étaient la principale source d'infection, que ce soit pendant l'élevage, le transport et l'attente. Ces résultats soulignent l'importance d'améliorer l'efficacité des protocoles de nettoyage et de désinfection. La faune sauvage semble également impliquée dans l'épidémiologie de Salmonella dans la filière porcine. Une forte similarité génétique a été observée entre les souches isolées de rongeurs et de cafards et celles isolées des porcs. Par ailleurs, une prévalence en Salmonella de 16,5% dans les saucisses à base de porc démontre que Salmonella dans la filière porcine peut représenter un risque pour la santé humaine à La Réunion. Ces travaux de recherche démontrent la nécessité d'agir aux différents maillons de la filière porcine. En outre, une lutte globale doit être envisagée, incluant les différentes filières, les industriels, les services vétérinaires et les pouvoirs publics. / Salmonella is a zoonotic bacterium that represents a public health burden all over the world. Pig is considered as one of the main reservoir of this pathogen. The goal of this research project was to identify the main measures that would limit the presence of Salmonella in the Reunionese pig production. Nearly 80% of the pig farms investigated were infected with Salmonella at the end of the fattening period. This asymptomatic infection was associated with several risk factors: geo-climatic conditions (altitude and rainfall), poultry production on the same site, the size of the farm and the presence of residual Salmonella in rooms before the loading of the pig batch. Furthermore, the longitudinal investigation highlighted that environmental residual Salmonella were the main source of infection whether at farm, during the transport and the lairage of pigs. These results strengthen the importance of improving efficiency of cleaning and disinfection procedures. The wild fauna of Reunion Island seems to be involved in the epidemiology of Salmonella in the pig production, since high genetically relationships were observed between Salmonella strains isolated from rodents and cockroaches and those isolated from pigs. Otherwise, our study also highlighted Salmonella prevalence of 16.5% in pork sausages demonstrating that Salmonella in the pig production may represent a risk for public health in Reunion Island. These works stress the need to act at each stage of the pig production. Moreover, a global effort has to be considered, including professionals of the different animal production, industrials, veterinary and health services and authorities.

Les sols sont le plus grand réservoir de carbone des écosystèmes terrestres, et la minéralisation des matières organiques par l’activité microbienne représente la majeure partie des flux de CO2 émis à la surface des continents.Dans ce travail, nous avons étudié les matières organiques extraites à l’eau (WEOM), qui correspondent à la fraction la plus réactive des matières organiques du sol (MOS). Nos objectifs étaient (i) d’identifier les liens de la dynamique du WEOM avec les communautés bactériennes, et avec les paramètres physico-chimiques du sol ; (ii) de réaliser une caractérisation chimique précise du WEOM.Il existe un lien fort entre la solubilité des MOS et les structures des communautés bactériennes, et une baisse de leur diversité impacte la dynamique des MOS et du WEOM, et provoque une baisse de la minéralisation des matières organiques. Une étude à l’échelle régionale a également permis d’identifier que les taux de MOS et d’argile contrôlent les quantités de WEOM et leur aromaticité. La caractérisation au niveau moléculaire a montré la présence d’un grand nombre de molécules ubiquistes dans le WEOM. À partir de ces analyses, nous avons également pu décrire les effets du couvert végétal et des propriétés physico-chimiques des sols sur la composition chimique du WEOM. / Soils are the greatest reservoir of C on the continents, and organic matter mineralisation bymicrobial activity represents the major part of the CO2 emitted by terrestrial ecosystems.In this work, we studied water-extractable organic matter (WEOM), which corresponds to themore reactive fraction of soil organic matter (SOM). Our objectives were (i) to identify therelationships of WEOM dynamics with bacterial communities, and with soil physico-chemicalparameters; (ii) to provide a precise chemical characterisation of WEOM.There is a strong link between SOM solubility and the structure of bacterial communities, andan erosion of their diversity has an impact on SOM and WEOM dynamics, and leads to adecrease in organic matter mineralisation. A study at the regional scale then allowed us to identifythat the SOM and clay contents control the quantities of WEOM and its aromaticity. TheWEOM characterisation at the molecular level revealed the presence of a large number ofubiquitous molecules in the WEOM. Based on these analyses, we were also able to describe theeffects of vegetation and soil physico-chemical properties on the chemical composition ofWEOM.