Bacillus thuringiensis sérotype israelensis (Bti) produit quatre toxines entomocides utilisées à grande échelle pour le biocontrôle des populations de diptères nuisibles et vecteurs de maladies : Cry4Aa, Cry4Ba, Cry11Aa et Cyt1Aa. Chacune de ces toxines présente un effet létal sur différents insectes mais, lorsqu’elles sont combinées, on observe un effet synergique et l’absence de résistance. Bien que cette synergie soit bien documentée par des tests de toxicité, il existe très peu d’information sur son mécanisme aux niveaux cellulaire et moléculaire. À l’aide d’intestins isolés des larves du moustique Aedes aegypti, le principal vecteur du paludisme, et de microélectrodes, nous avons observé une dépolarisation membranaire en présence de Cyt1Aa et de Cry4Aa individuellement. Cette dépolarisation se produit cependant plus rapidement lorsque la Cyt1Aa est utilisée en même temps que la Cry4Aa. D’autre part, des expériences réalisées avec la sonde calcique Fura-2 sur une lignée cellulaire provenant d’Anopheles gambiae (Ag55), ont révélé une forte activité lytique de la Cyt1Aa, mais très peu d’effets des autres Cry, et ce même en combinaison. Nous avons dissocié les cellules de l’épithélium intestinal isolé du moustique pour des expériences de Fura2. Nos résultats, quoique préliminaires, montrent les effets variables de ces toxines lorsqu’elles sont administrées seules sur les cellules dissociées : une augmentation du calcium intracellulaire, ou une fuite de la sonde se traduisant par une perte du signal fluorescent, ou la lyse cellulaire. On observe également en présence de Cyt1Aa et de Cry4Ba, que les effets sont presque instantanés. / Bacillus thuringiensis var israelensis (Bti) produces four insecticidal toxins used around the world to control disease-borne and harmful dipterans populations: Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. They each present their lethal effect on different dipterans, but combined, they generate a synergistic activity and a reduced resistance is observed. Though these synergies are well documented and supported by toxicity bioassays, little is known regarding the cellular and molecular mechanisms of these synergies. Here, by using freshly isolated midguts from the mosquito Aedes aegypti, an important malaria vector, and glass microelectrodes, we measured the electrical potential of the apical membrane when exposed to these toxins alone or in combination. We observed a depolarisation when treated with Cyt1Aa and Cry4Aa. Toxin mixture assays only revealed a faster depolarisation of the membrane when the above two toxins were combined together, and a variety of responses with other toxin mixtures. Microspectrofluometry using the calcium probe Fura-2 on an immortal cell line from Anopheles gambiae (Ag55) showed massive effect of Cyt1Aa, but very little effect of the Cry toxins alone or in mixture. Microspectrofluometry experiments were also conducted on freshly dissociated cells from Aedes aegypti. Though these experiments are innovative and the results preliminary, it was observed that some cells responded differently to Cyt1Aa and Cry4Ba, showing the various ways these toxins affect cells, by inducing either intracellular calcium change, or by entirely losing the probe, or by cell lysis. The mixture of these toxins is very efficient and almost instantaneous.

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%.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-21T12:25:29Z No. of bitstreams: 1 Andrea Maria Andre Gomes.pdf: 1093534 bytes, checksum: b089cef12b68df384d0aec41d274408e (MD5) / Made available in DSpace on 2017-02-21T12:25:29Z (GMT). No. of bitstreams: 1 Andrea Maria Andre Gomes.pdf: 1093534 bytes, checksum: b089cef12b68df384d0aec41d274408e (MD5) Previous issue date: 2003-03-14 / Lettuce (Lactuca sativa L.) is one of the most consumed vegetables in the world. In spite of the importance of this crop in Pernambuco Brazil, there are no studies on the use of plant growth-promoting rhizobacteria for production of organic transplants, as well as a diagrammatic scale for evaluating the severity of Cercospora leaf spot (Cercospora longissima) in epidemiological surveys. Epiphytic and endophytic bacterial strains applied through simultaneous bacterization on seedlings and plants were tested in greenhouse using cv. Verônica. In field the strains C25 (Bacillus thuringiensis subvar. kenyae) and C116 (Bacillus pumilus) were tested separately and in mixture using cvs. Verdinha and Verônica. Seedlings treated with C116 showed significant increase in relation to controls for root fresh weight, shoot fresh weight and total fresh weight as well as those treated with C25 for root fresh weight and total fresh weight. The strain C25 also significantly increased the level of foliar N. Aiming tostandardize methods to quantify the severity of Cercospora leaf spot of lettuce, a diagrammatic scale was developed with 1, 3, 6, 11, 20, 34, 51 and 68% diseased leaf areas. The diagrammatic scale was validated by 10 raters using 50 leaves with different levels of severity, previously measured by the software AutoCAD® R14 for Windows. There were performed two evaluations with a 7-day interval in which different sequences of the same leaves were visually estimated by the same raters. The accuracy and precision of each rater was determined through simple linear regression between actual and estimated severity. The scale proportioned good levels of accuracy and precision, and despite severity tended to be underestimated, the absolute errors were around 10%. Generally the raters showed good repeatability of estimative. Surveys ofCercospora leaf spot severity were performed in commercial plantations, including conventional and organic systems, in Mata and Agreste of Pernambuco, in two planting seasons. Disease prevalence in conventional systems ranged from 92 to 100% and in organic systems from 88 to 100%. In conventional plantings the severity significantly varied from 0.84 to 16.86 in January-April and 0.00 to 2.44 in June-September. In this system significant differences were observed among the counties Chã Grande (4.99) and Vitória de Santo Antão (9.14), only in January-April. Still in the conventional farming, there were no significant differences of disease severity among groups, cultivars of lettuce and number of fungicide applications within each season. In the organic system disease severity varied significantly from 0.07 to 22.53 in January-April and from 0.00 to 15.18 in June-September. In this system there were no significant differences fordisease severity averages in Bom Jardim, Chã Grande, Glória de Goitá and Gravatá, within each season. In January-April, but not in June-September, the average of Cercospora leaf spot severity significantly differed among groups and cultivars of lettuce. The group Lisa cv. Verdinha (0,68), showed significantly less severity than the groups Crespa cv. Verônica (10.61) and Americana cv. Lucy Brown (7.71). The Pearson test did not find significant correlations for severity between the studied seasons in both conventional and organic systems. The Kruskall-Wallis test did not detected significant differences for severity among the conventional and organic systems in each period. However there was significant difference for severity between the two seasons in each system. / A alface (Lactuca sativa L.) é uma das hortaliças mais consumidas no mundo. Apesar da importância desta cultura em Pernambuco, inexistem estudos sobre o uso de bactérias promotoras de crescimento de plantas para a produção de mudas orgânicas bem como de uma escala diagramática para a avaliação da severidade da cercosporiose (Cercospora longissima) em levantamentos epidemiológicos. Isolados bacterianos epifíticos e endofíticos, aplicados por bacterização simultânea nas sementes e substrato, foram testados em estufa utilizando a cultivar Verônica. Em campo, foram testados os isolados C25 (Bacillus thuringiensis subvar. kenyae) e C116 (Bacillus pumilus), separadamente e em mistura, nas cultivares Verdinha e Verônica. As mudas apresentaram aumento significativo para matéria fresca da raiz, da parte aérea e totalutilizando o isolado C116 e para matéria fresca da raiz e total utilizando-se o C25. O isolado C25 elevou significativamente o teor foliar de N. Para padronizar métodos de quantificação da severidade da cercosporiose, foi desenvolvida uma escala diagramática com os níveis 1, 3, 6, 11, 20, 34, 51 e 68% de área foliar lesionada. A escala diagramática foi validada por 10 avaliadores que utilizaram 50 folhas com sintomas da doença em diferentes níveis de severidade, mensuradas pelo programa AutoCAD® R14 for Windows. Realizou-se duas avaliações com intervalo de sete dias, onde seqüências diferentes das mesmas folhas foram estimadas visualmente pelos mesmos avaliadores. A acurácia e a precisão de cada avaliador foram determinadas por regressão linear simples, entre a severidade real e a estimada. A escala proporcionou bons níveis de acurácia e precisão e, apesar de evidenciar tendência a subestimativa, os erros absolutosconcentraram-se na faixa de 10%. Em geral, os avaliadores demonstraram boarepetibilidade nas estimativas. Foram efetuados levantamentos da severidade da doença em áreas de cultivo convencional e orgânico da Zona da Mata e Agreste de Pernambuco, em dois períodos de plantio. Foi registrada prevalência da doença em cultivo convencional variando de 92 a 100% e em orgânico, de 88 a 100%. No cultivo convencional, a severidade variou significativamente entre 0,84 a 16,86 no período janeiro-abril e 0,00 a 2,44 no período junho-setembro. Nesse cultivo, foram constatadas diferenças significativas entre os municípios Chã Grande (4,99) e Vitória de Santo Antão (9,14), apenas no período de janeiro-abril. Ainda no cultivo convencional, não foram constatadas diferenças significativas da severidade da cercosporiose entre grupos e cultivares de alface e, entre número de aplicações de fungicidas, dentro de cada período. No cultivo orgânico, verificou-se diferença significativa na severidade da doença em janeiro-abril variando de 0,07 a 22,53 e em junho-setembro variando de 0,00 a 15,18. Nesse cultivo não foram constadas diferenças significativas para médias de severidade nos plantios de Bom Jardim, Chã Grande, Glória de Goitá e Gravatá, dentro de cada período. No período janeiro-abril, mas não em junho-setembro, as médias de severidade da cercosporiose diferiram significativamente entre grupos e cultivares de alface. O grupo Lisa, cv. Verdinha (0,68), diferiu, significativamente dos grupos Crespa cv. Verônica (10,61) e Americana cv. Lucy Brown (7,71). Não foram constatadas correlações significativas para a severidade entre os períodos estudados, tanto no cultivo convencional quanto no orgânico. Pelo teste de Kruskall-Wallis, não houve diferença significativa para a severidade entre os cultivos convencional e orgânico, nos dois períodos. Por outro lado, houve diferença significativa para a severidade entre os dois períodos nos dois tipos de cultivo.

Orientadora: Janete Apparecida Desidério Sena / Banca: Manoel Victor Franco Lemos / Banca: Cristina Lacerda Soares Petrarolha Silva / Banca: Gislayne Fernandes Lemes Trindade Vilas-Bôas / Banca: Odair Aparecido Fernandes / Resumo: Aedes aegypti (Linnaeus), principal vetor da dengue no Brasil, tem sido combatido com o uso maciço de produtos químicos, contribuindo com o desenvolvimento de resistência e inviabilizando o controle do inseto. Bioinseticidas à base de Bacillus thuringiensis vêm apresentando resultados satisfatórios no controle de dípteros, devido à produção de proteínas bioinseticidas denominadas Cry (cristal), Cyt (citolíticas) e Chi (quitinase) e os efeitos sinergísticos existentes entre elas. O presente trabalho objetivou a seleção de isolados de B. thuringiensis, portadores de genes cry, cyt e chi com alta eficiência no controle de A. aegypti. Uma coleção de 1073 isolados de B. thuringiensis, provenientes de diversas regiões brasileiras, foi submetida à Reação em Cadeia da Polimerase (PCR), com base nos iniciadores específicos para os genes cry4Aa, cry4Ba, cry10Aa, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa e chi. Os isolados dípteros-específico foram avaliados quanto à estruturação genética e através de bioensaios seletivos e quantitativos, para determinação da CL50 e CL90. Apenas 45 (4,19%) apresentaram amplificação para os genes cry e cyt e destes, 25 (54,34%) eram quitinolíticos. Foram definidos 21 haplótipos entre os 45 isolados de B. thuringiensis, os quais foram submetidos aos bioensaios seletivos, indicando 13 isolados que causaram 100% de mortalidade às larvas de A. aegypti. Os bioensaios quantitativos e análise de agrupamento permitiram selecionar quatro isolados altamente tóxicos às larvas de A. aegypti. A identificação dos genes cry, cyt e chi de B. thuringiensis e análise dos efeitos sinergísticos entre as toxinas, associadas à análise da toxicidade a insetos vetores permitiram a seleção de isolados que poderão ser utilizados em formulações de novos bioinseticidas brasileiros, podendo contornar possíveis problemas de resistência. / Abstract: Aedes aegypti (Linnaeus), the main vector of dengue fever in Brazil, has been controlled with massive use of chemical products wich, contributed to the development of resistance decreasing the insect control efficacy. Bioinsecticides based on Bacillus thuringiensis is currently presenting satisfactory results on controlling dipterans, due to the production of Cry proteins (crystal proteins), Cyt (citolitic) and Chi (chitinase) with synergistic effects between them. The present work aimed to select B. thuringiensis isolates with cry, cyt and chi genes exhibiting high efficiency on A. aegypti larvae. A bacterial collection containing 1073 isolates of B. thuringiensis, obtained from different Brazilian locations, had their DNA isolated and submitted to PCR amplifications using specific primers for the genes cry4Aa, cry4Ba, cry10Aa, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa e chi. The dipterans-especific isolates were then evaluated for the genetic structure and through selective and latter to quantitative bioassays for the determination of LC50 and LC90. Forty five out of 1073 isolates (4.19%) presented amplicons for the cry and cyt genes and from these 25 (54.34%) were detected as chitinolitic. Twenty one haplotypes were identified among the 45 isolates of B. thuringiensis, which were submitted to the selective bioassays, indicating 13 isolates that caused 100% mortality of A. aegypti larvae. The quantitative bioassays and the statistical analysis have pointed four toxic isolates to A. aegypti larvae. The identification of the cry, cyt and chi genes of B. thuringiensis and the analysis of the synergistic effects among the toxins, along with the toxicity analysis towards the vector insects allowed the selection of a set of isolates that can be used for the formulation of new bioinsecticides from Brazilian origin, that would avoid or at least delay the appearance of resistance problems. / Doutor

Publicación a texto completo no autorizada por el autor / Evalúa y selecciona un agente fotoprotector y un agente fagoestimulante que permita mejorar la actividad entomotóxica de B. thuringiensis sobre larvas de S. frugiperda. Para ello, se evaluó el espectro de absorbancia de 9 agentes fotoprotectores, la compatibilidad con B. thuringiensis y además de exposición a la luz artificial del sol (UV A y UVB); seleccionándose al Ac. Ascórbico como el mejor agente fotoprotector compatible con B. thuringiensis. Para la selección del fagoestimulante se realizaron pruebas de preferencia alimenticia con larvas neonatas de Spodoptera frugiperda así como la evaluación de la compatibilidad con B. thuringiensis seleccionándose la hoja de maíz tratada. Se evaluó mediante bioensayos la efectividad de los agentes seleccionados observándose una disminución del DL50 cuando se adicionaron fagoestimulante y fotoprotector a la cepa comparándose con la cepa sola y sometiéndola a radiación UV durante 4 y 8 horas. / Tesis

La résistance aux insecticides chez les moustiques pose des problèmes de santé publique car ils sont vecteurs de nombreuses maladies. Une alternative aux insecticides chimiques est l'utilisation du bactério-insecticide Bacillus thuringiensis subsp israelensis (Bti) qui a l'avantage de produire un mélange de six toxines spécifiques des Diptères. Cependant le Bti commercial peut proliférer et s'accumuler, entrainant une forte toxicité dans les litières végétales de certains gîtes à moustiques. Afin d'étudier l'évolution de la résistance au Bti chez les moustiques, j'ai sélectionné en laboratoire une souche d'Aedes aegypti avec des litières végétales contenant des toxines de Bti. Une résistance multigénique aux toxines Cry du Bti est apparue en seulement quelques générations chez la souche sélectionnée. Plusieurs approches ont été utilisées pour rechercher les bases génétiques de la résistance au Bti chez la souche d'Ae. aegypti résistante. Deux "scans génomiques" ont permis de déterminer plusieurs régions du génome présentant des signatures de sélection. Ensuite, les niveaux de transcription de plus de 6000 gènes ont été étudiés par séquençage haut débit. La combinaison de ces résultats avec une approche "gènes candidats" a permis d'obtenir une liste de gènes potentiellement liés à la résistance au Bti. Parmi les gènes identifiés, un gène codant pour une cadhérine présente des signatures de sélection chez la souche résistante et semble donc impliqué dans la résistance au Bti. De plus, une étude de génomique des populations de l'espèce de terrain Aedes rusticus traitée depuis 20 ans au Bti a mis en évidence des signatures de sélection liées au traitement et des flux de gènes importants chez cette espèce dans la région Rhône-Alpes. La caractérisation de facteurs génétiques liés à la résistance et de facteurs biologiques liés aux espèces traitées peut aider à la mise en place de stratégies de gestion limitant l'évolution de la résistance au Bti dans ces populations.

No description available.

Infection of small ruminants by gastrointestinal nematodes (GIN) is a major health concern because they cause substantial economic losses, especially in the tropics and sub-tropics. For many years, control of GIN has been based upon use of anthelmintics. However, there is now a global challange because mutant GIN individuals can tolerate most of the widely used anthelmintics. Therefore, alternative control measures are needed. The objective of the study was to screen a number of plant species for their anthelmintic effects, and to evaluate selected strains of Bacillus thuringiensis (Berliner) and Clonostachys rosea (Schroers) for activity against sheep GIN. Subsequently, the combined treatments would test a dual control strategy for nematodes by using a combination of plant extracts with biocontrol agents. Ethanol extracts of 25 plant species were screened for their anthelmintic effects against Haemonchus contortus (Rudolphi 1803). Extracts of each plant were used in vitro at various concentrations (10, 20 and 30%) to treat 10 day faecal cultures. Five plants with high efficacies (Ananas comosus L. Merr., Aloe ferox Mill., Allium sativum Linn., Lespedeza cuneata Dum. Cours. and Warburgia salutaris Bertol.f. Chiov) were selected for further investigation, using ethanol, dichloromethane and water extracts at four concentrations (2.5, 5, 10 and 20%). Ethanol was the most effective solvent. Larval counts decreased as a result of increasing extract concentrations. An ethanolic extract of Lespedeza cuneata caused more than 70% mortality at all concentrations. In an in vivo study, the five plants A. comosus, A. ferox, A. sativum, L. cuneata and W. salutaris extracts were compared to a positive Control (Equimax®, a modern anthelmintic based on abamectin and praziquantel). Gender, eggs count (EPG₀) and initial body weights were used in assigning sheep (24 females and 24 males) to six groups. Each group was randomly assigned a treatment. Plant extracts were applied as an oral dose (100 mg kg⁻¹ BW), one dose per week per animal for 42 days (Phase 1). Subsequently, the same sheep were dosed for three consecutive days with the same treatments, keeping them in the same groups (Phase 2). Rectal faecal samples were taken for counting of eggs per gram of faeces (EPG) and L₃ larvae per gram (LPG) in faecal cultures. With application of plant extracts, the EPG count decreased with time (P<0.001), and the impact of the plant extracts increased (P<0.001) with time. Two extracts, from A. comosus and L. cuneata, were the most effective in Phase 1 (58% and 61% reduction of EPG, respectively,), and in Phase 2 (77% and 81% reduction of EPG, respectively). In a study on potential biocontrol agents, two strains of Bacillus thuringiensis (Bt) and one of Clonostachys rosea f. rosea (C. rosea), and compared with a diatomaceous earth (DE) product for their anthelmintic activity in sheep. Bacillus thuringiensis and C. rosea were fed to sheep at a rate of 1g kg⁻¹ BW, and DE was fed at 2% of sheep diet. The biocontrol treatments had no effect on EPG (P>0.05), but reduced GIN larvae per gram (LPG) (P<0.001) in faecal culture. Efficacy varied with time (P<0.001). By Day 7 Bt, C. rosea and DE had caused mortalities of GIN of 75.7, 86.9 and 60.6%, respectively. In addition, the efficacy of feeding 1g kg⁻¹ BW of C. rosea chlamydospores to sheep every day, every second day and every third day was tested. Daily feeding of fungal chlamydospores reduced LPG (a count of 12±1.67 GIN larvae) (P<0.001) more than feeding them the biocontrol agent every second day (39±0.77) or third day (58±1.77). By Day 12, feeding the biocontrol agent to sheep every day, every second day, or every third day caused mortality of GIN larvae of 90, 63 and 49%, respectively. Four dietary levels (treatments) of C. rosea (0.25g (F1), 0.5g (F2), 1g (F3) and control (C) of C. rosea product kg-1 BW) were tested. Treatments were each mixed with a complete diet and fed to sheep once daily for 10 weeks, according to body weights. Increased doses of the biocontrol agent reduced LPG (P<0.001), larval development (LD) (P<0.001), and increased efficacy (P<0.001). On Day 70, F1, F2, F3 and the Control controlled LD by 33.3, 72.3, 89.4 and 2.6%, respectively. Clonostachys rosea was effective in reducing third stage larvae (L₃) on pastures significantly (P<0.001) by Day 63 and Day 70. Ethanolic extracts of A. comosus, A. ferox, A. sativum, L. cuneata and W. salutaris all reduced egg production by GIN parasites of sheep. Feeding sheep cultured chlamydospores of a biocontrol fungus, Clonostachys rosea, reduced counts of nematode larvae in sheep; and 1g C. rosea chlamydospores kg⁻¹ BW daily was enough to reduce nematode infective larvae, therefore reducing the degree of pasture contamination. An initial trial showed that the combination of the two treatments of an A. comosus extract and C. rosea chlamydospores was more effective than either treatment on its own in controlling gastrointestinal nematodes in sheep. A long-term trial is being undertaken currently to confirm this finding. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.