Clostridium perfringens (C. perfringens) causes diseases such as necrotic enteritis in poultry. Current methods for early detection of C. perfringens, including culture, and molecular techniques (PCR and qPCR), have logistical limitations in detecting pathogens on-site because they require specialized equipment and long sample preparation time. This study describes developing a highly sensitive on-site LAMP assay combined with a simple DNA extraction method to detect C. perfringens. Results showed that the LAMP assay was highly specific and sensitive with the optimal reaction conditions: 63°C for 30 mins with primer concentration of 1.2 mM FIP/BIP and 0.2 mM F3/B3. The HotSHOT method was the most convenient DNA extraction method for the on-site LAMP assay with respect to the processing time, cost, and minimal equipment requirement. Therefore, the HotSHOT DNA extraction method followed by the LAMP assay could serve as a rapid on-site molecular test for C. perfringens in the field.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Made available in DSpace on 2017-07-25T19:29:42Z (GMT). No. of bitstreams: 1 Hellen Aparecida Arantes.pdf: 691625 bytes, checksum: a3ecb17b706b52a2674dc29e0a5e82cc (MD5) Previous issue date: 2008-12-15 / The development and grain yield of the crop of wheat are influenced by several factors, among which is the interference caused by pathogens present in crops. Currently, the main tool for controlling these pathogens in crops is the use of constant application of fungicides. However, this practice is causing pathogens resistant to these groups of fungicides used, as well as encouraging the expression of the mutagenic character of these populations. In an attempt to provide more tools to control the diseases of wheat to the farmer, this study aimed to: a) assess the effect of timing of implementation and sources of phosphite and acibenzolar-S-methyl alone and combined with the fungicide pyraclostrobin + epoxiconazole in control the powdery mildew (Blumeria graminis f. sp. tritici) and the leaf rust (Puccinia triticina), b) assess the effect of different rates, sources of phosphite and acibenzolar-S-methyl isolated and associated to the fungicide pyraclostrobin + epoxiconazole in control of the spots brown (Bipolaris sorokiniana) and yellow (Drechslera tritici-repentis); c) and in vitro effect of different concentrations of phosphite and pyraclostrobin + epoxiconazole on the mycelial growth and germination of conidia of Bipolaris sorokiniana. About the control of powdery mildew and rust can be concluded that the phosphites have no effect on these pathogens and do not change their productivity. It is also clear that there is no difference between the different sources of phosphites to control the powdery mildew and leaf rust, which occurs on powdery mildew and leaf rust's only when the phosphite associated with the fungicide is applied. About the control of yellow and brown stains concluded that the dose of 1048 g i.a. ha-1 potassium phosphite reduces the severity of leaf spots and can be used as a tool in the management of leaf spots. In in vitro tests concluded that the phosphites have direct action on Bipolaris sorokiniana. / O desenvolvimento e o rendimento de grãos da cultura do trigo são influenciados por diversos fatores, entre os quais se encontram a interferência causada por patógenos presentes nas lavouras. Atualmente, a principal ferramenta de controle destes patógenos nas lavouras é o uso de constantes aplicações de fungicidas. Contudo, essa prática vem ocasionando resistência destes patógenos aos grupos de fungicidas utilizados, bem como favorecendo a expressão do caráter mutagênico destas populações. Na tentativa de disponibilizar mais uma ferramenta de controle às doenças do trigo ao agricultor, este trabalho objetivou: a) avaliar o efeito de épocas de aplicação e de fontes de fosfito e acibenzolar-S-metil isolados e combinados com o fungicida epoxiconazole + piraclostrobina no controle do oídio (Blumeria graminis f. sp. tritici) e da ferrugem da folha (Puccinia triticina); b) avaliar o efeito de diferentes doses, fontes de fosfito e acibenzolar-S-metil isolados e associados ao fungicida epoxiconazole + piraclostrobina no controle das manchas marrom (Bipolaris sorokiniana) e mancha amarela (Drechslera tritici-repentis); c) e efeito in vitro de diferentes concentrações de fosfito e epoxiconazole + piraclostrobina sobre o crescimento micelial e a germinação de conídios de Bipolaris sorokiniana. Sobre o controle de oídio e ferrugem pode-se constatar que os fosfitos não possuem efeito sobre estes patógenos e não alteram a produtividade. Concluiu-se ainda que não existe diferença entre as diferentes fontes de fosfitos no controle de oídio e ferrugem da folha, que ocorre efeito sobre o oídio e a ferrugem da folha apenas quando o fosfito é aplicado associado ao fungicida. Sobre o controle das manchas amarela e marrom conclui-se que a dose de 1048 g i.a. ha-1 de fosfito potássico reduz a severidade das manchas foliares, podendo ser utilizado como uma ferramenta no manejo das manchas foliares. Nos testes in vitro conclui-se que os fosfitos possuem ação direta sobre Bipolaris sorokiniana.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Travail en codirection avec M. Frédéric Raymond / Avec la présente augmentation de la résistance aux antibiotiques et l’inquiétude des consommateurs, de nouvelles alternatives sont nécessaires afin de contrôler l’entérite nécrotique (EN), une maladie causant des millions de dollars en pertes économiques pour l’industrie de la volaille mondialement. Les bactériocines sont des peptides antimicrobiens produits par une bactérie pour tuer ou inhiber la croissance d’autres compétiteurs bactériens. La perfrine est la seule bactériocine reportée associée aux souches pathogènes de Clostridium perfringens, l’agent causal de l’EN. Les objectifs de cette étude étaient de détecter les souches commensales de Clostridium perfringens possédant une activité antimicrobienne contre des souches pathogènes de C. perfringens et d’identifier et caractériser les bactériocines produites. Les souches commensales de C. perfringens ont été sélectionnées à partir de notre banque de souches. L’activité antimicrobienne de ces souches a été testée contre des souches pathogènes de C. perfringens en utilisant la méthode d’inhibition sur gélose. Une souche commensale active démontrant une activité antimicrobienne a été cultivée et ses bactériocines ont été partiellement purifiées grâce à la précipitation au sulfate d’ammonium et par chromatographie (HPLC). À la suite de chaque chromatographie, l’activité antimicrobienne des fractions a été vérifiée en utilisant la méthode décrite précédemment afin de choisir les fractions contenant les bactériocines. La susceptibilité enzymatique, la stabilité à la chaleur et au pH et le poids moléculaire estimé des bactériocines ont été caractérisés. Les bactériocines étudiées étaient sensibles à la protéinase K, thermolabiles, stables à pH entre 4 et 8 et leur poids moléculaire étaient supérieur à 30 kDa. Le génome de la souche CP1676 a été séquencé et des analyses bio-informatiques ont été réalisées. Nous avons trouvé 28 séquences de bactériocines potentielles, mais seulement 4 d’entre elles semblaient être prometteuses. Dans cette étude, des souches commensales de C. perfringens produisant des bactériocines actives contre des souches pathogènes ont été identifiées. Ces bactériocines pourraient devenir une alternative intéressante aux antibiotiques afin de contrôler l’entérite nécrotique, mais davantage d’information est nécessaire. / With the current increase in antimicrobial resistance and consumers’ concern, new alternatives are needed to control necrotic enteritis (NE), a disease that causes billions of dollars in economic losses to the poultry industry worldwide. Bacteriocins are antimicrobial peptides produced by bacteria to kill or inhibit the growth of other bacterial competitors. Perfrin is the only reported bacteriocin associated with pathogenic strains of Clostridium perfringens, the causal agent of NE. The aims of this study were to screen for commensal Clostridium perfringens strains with an antimicrobial activity against C. perfringens pathogenic strains and to identify and characterize the produced bacteriocins. Commensal C. perfringens strains were selected from our bacterial collection. Antimicrobial activity of those selected strains was tested against C. perfringens pathogenic strains using the agar spot test method. An active commensal strain showing antimicrobial activity was cultured and its bacteriocins were partially purified using the ammonium sulfate precipitation method and HPLC. After each chromatography, antimicrobial activity of fractions was tested using the method described above to choose fractions containing bacteriocins. Enzyme susceptibility, heat and pH stability and the estimated molecular weight of the bacteriocins were characterized. The studied bacteriocins were sensitive to proteinase K, thermolabile, stable at pH between 4 and 8 and their molecular weight higher than 30 kDa. CP1676 strain genome was sequenced and bioinformatics were performed. We found 28 potential bacteriocin sequences, but 4 of them seemed to be promising. In this study, commensal C. perfringens strains producing bacteriocins active against pathogenic strains have been identified. These bacteriocins could be an interesting alternative to antibiotics for the control of necrotic enteritis but further data is still needed.

Avian pathogenic Escherichia coli and Clostridium perfringens cause economic and welfare concerns to the broiler industry. The recent shift to no antibiotics ever (NAE) production has increased disease incidence. The objectives of this study were to determine the influence of season, age of flock, and sample type on E. coli prevalence and virulence and to identify C. perfringens prevalence and toxinotypes in NAE farms. Results indicated high prevalence of virulent E. coli; prevalence of virulent E. coli decreased from Spring to Summer. Virulent E. coli showed high resistance to antimicrobials. Serogroups O8 and O78 were most prevalent in virulent E. coli. C. perfringens prevalence was very low and all recovered isolates were toxinotype A with variation in netB, cpb2, and tpeL presence. In conclusion, NAE farms should have measures to control E. coli infections, especially in spring season. Further studies are required to confirm the lower prevalence of C. perfringens.

Syftet med denna masterstudie var att jämföra den smärtlindrande effekten av fullständig utrensning och desinfektion av rotkanalsystemet med enbart utrymning av pulpakavum, utan instrumentering av rotkanalerna, som akutbehandling hos tänder med symtomatisk apikal parodontit. Materialet utgjordes av 16 patienter insamlade på Tandvårdshögskolans jourmottagning. Patienter med svullnad och allmänpåverkan exkluderades. Patienterna valdes slumpmässigt ut till att genomgå någon av behandlingarna. Den preoperativa smärtnivån och ev. analgetikaintag registrerades. Tre till fem dagar efter utförd behandling kontaktades patienterna per telefon och fick då gradera sin postoperativa smärtnivå samt analgetika- och/eller antibiotikaintag. Resultatet visade att 100% av patienterna som behandlades med fullständig utrensning erhöll tillfredställande smärtlindring jämfört med 71% av de patienter som behandlades med enbart utrymning av pulpakavum. Skillnaden var inte statistiskt signifikant. 44% av patienterna som behandlades med fullständig utrensning hade tagit smärtstillande postoperativt medan 29% av patienterna i utrymningsgruppen hade gjort det. Skillnaden var inte signifikant. Ingen av patienterna oavsett behandling hade tagit antibiotika efter behandlingen. Ingen skillnad förelåg i andelen patienter som blev tillräckligt smärtlindrade mellan fullständig utrensning och enbart utrymning av pulpakavum hos tänder med symtomatisk apikal parodontit. Det var inte heller någon skillnad i smärtsänkning mellan de två behandlingarna. / The aim was to compare the pain relieving effect of complete chemo mechanical disinfection of the root canal system with removal of necrotic tissue in the pulp chamber without instrumentation of the root canals as emergency treatment in teeth with symptomatic apical periodontitis. The material consisted of 16 patients collected at the emergency clinic at Malmö University. Patients with swelling and/or systemic involvement were excluded. The patients were randomized to either treatment. The preoperative pain level and intake of analgesics was registered. Three till 5 days postoperatively the patients were contacted and asked to grade the current pain level and intake of analgesics and/or antibiotics. The results showed that 100% of the patients treated with complete chemo mechanical disinfection of the root canal system obtained satisfying pain relief compared to 71% for patients treated with removal of necrotic tissue in the pulp chamber. 44% of the patients treated with chemo mechanical disinfection of the root canal system had some kind of analgesics postoperatively compared to 29% for those treated with removal of necrotic tissue in the pulp chamber. No patients in either group reported use of antibiotics postoperatively. The conclusion was that both complete chemo mechanical disinfection and removal of necrotic tissue implied a significant pain relief as emergency treatment in teeth with symptomatic apical periodontitis. There was no difference between the two treatments concerning the number of patients who obtained sufficient pain relief or in pain relieving effect.

The cytosolic protein rubicon (RUBCN) has been implicated in the removal of necrotic debris and autoimmunity. However, the role of RUBCN in models of acute kidney injury (AKI), a condition that typically involves necrotic kidney tubules, was not investigated. Here, we demonstrate that RUBCN-deficient mice are hypersensitive to renal damage induced by ischemia-reperfusion injury (IRI) and cisplatin-induced AKI. Combined deficiency of RUBCN and mixed lineage kinase domain-like (MLKL) partially reversed the sensitivity in the IRI model suggesting that the absence of RUBCN sensitizes to necroptosis in that model. Necroptosis is known to contribute to TNFα-induced severe inflammatory response syndrome (SIRS), but we detected no statistically significant difference in overall survival following injection of TNFα in RUBCN-deficient mice. We additionally generated RUBCN-deficient mice which lack gasdermin D (GSDMD), the terminal mediator of pyroptosis, but no reversal of the AKI phenotype was observed. Finally, and in contrast to the previous understanding of the role of RUBCN, we did not find a significant autoimmune phenotype in RUBCN-deficient mice, but detected chronic kidney injury (CKD) in aged RUBCN-deficient mice of both sexes. In summary, our data indicate that RUBCN-deficient mice are hypersensitive to kidney injury.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25340号 / 農博第2606号 / 新制||農||1107(附属図書館) / 学位論文||R6||N5512 / DFAM / 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 寺内 良平, 教授 吉田 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Clostridium perfringens causa enterite necrótica aviária devido à produção de toxinas que lesam o intestino. Neste estudo, de 94 amostras nove apresentaram C. perfringens, totalizando 22 isolados. Todos exceto um isolado, possuíram os genes nanI (95%) e/ou nanJ (81%), e 19/22, mostraram atividade neuraminidase em hemácias de frango. A atividade hemaglutinante foi observada em poucos isolados (26%). Todos os isolados foram plc positivos (toxina α), sendo classificados como tipo A. Sete isolados (31,8%) abrigaram o gene tpeL que codifica a toxina TpeL. Isolados tpeL+ mostraram efeito citotóxico característico da ação desta toxina. Alguns isolados mostraram capacidade de aderir e invadir células Vero. A maioria dos isolados foi resistente à sulfaquinoxalina (100%), cefalexina (95%) e eritromicina (95%) e sensíveis (100%) à cefoxitina, amoxicilina, enrofloxacina, amoxicilina-ácido clavulânico, penicilina-estreptomicina, cloranfenicol e metronidazol. Todos os isolados foram agrupados geneticamente em sete clusters, apresentando se como um grupo heterogêneo. / Clostridium perfringens cause avian necrotic enteritis due to production of toxins that damage the intestine. In this study, nine out of 94 samples had C. perfringens, totaling 22 isolates. All the isolates with exception of one, possessed the genes nanI (95 %) and/or nanJ (81 %), and 19/22 showed neuraminidase activity in chicken erythrocytes. The hemagglutinating activity was observed in a few isolates (26 %). All isolates were plc positive (toxin α) being classified as type A. Seven isolates (31.8%) harbored tpeL gene encoding the toxin TpeL. TpeL + isolates showed characteristic cytotoxic effect of the action of this toxin. Some isolates showed ability to adhere and invade Hep-2 cells. Most of the isolates were resistant sulphaquinoxaline (100%), cephalexin (95%) and erythromycin (95%) and sensitivity (100%) to cefoxitin, amoxicillin, enrofloxacin, amoxicillin - clavulanic acid, penicillin -streptomycin, chloramphenicol and metronidazole. All isolates were genetically grouped into seven clusters, presenting itself as a heterogeneous group.