Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-05-18T18:09:32Z No. of bitstreams: 1 2015 - Bruno Oliveira de Carvalho.pdf: 1725115 bytes, checksum: 78e6b6f127705b066a8a0f75bbb08e88 (MD5) / Made available in DSpace on 2018-05-18T18:09:32Z (GMT). No. of bitstreams: 1 2015 - Bruno Oliveira de Carvalho.pdf: 1725115 bytes, checksum: 78e6b6f127705b066a8a0f75bbb08e88 (MD5) Previous issue date: 2015-03-17 / CAPES / The knowledge of bacterial genetic information has enable advances in many areas allowing detecting microorganisms and evaluating the production of virulence factors and resistance in certain populations. This knowledge allows the detection of microorganisms, as well as the evaluation of virulence and resistance factors production in certain populations. More recently, molecular diagnosis based on the detection of specific fragments from these agents has been implemented in no viable bacterial cultivation environments or hampered by biotic or abiotic conditions. Besides specific agent detection, bacterial diversity analysis might be an important criterion, such as assessing soil quality. Cultivation has been the most common way for evaluating bacterial diversity. However, most of soil bacteria are not cultivable; therefore, the employment of molecular tools has been increasingly used on this sort of analysis. This survey aimed standardizing the use of cultivation independent techniques for granting three agriculture sectors demands. The first chapter standardized the employment of DGGE technique for analyzing milk bacterial diversity from mastitis and healthy teats belonging to the same animal. Results pointed to differences on bacterial profile from both groups. The second chapter evaluated the impact of the employment of drill cuttings with castor beans and cramble pies association as agricultural fertilizers by DGGE analysis. The application of this technique was efficient for evaluating treatments, as well as, confirming the treatments contribution for increasing bacterial diversity in soil samples. The third chapter compared the cultivation technique sensitivity for American Foul Brood (AFB) diagnosis, a bee disease caused by Paenibacillus larvae, to cultivation independent technique by PCR. This survey demonstrated that sensitivity of the technique was greater than that one by P. larvae cultivation technique. Thus, the present survey concluded that cultivation independent techniques were useful for granting agriculture sectors demands. / O conhecimento das informa??es gen?ticas bacterianas tem permitido avan?os em diversas ?reas do conhecimento. Esse conhecimento permite detectar microrganismos, bem como avaliar a produ??o de fatores de virul?ncia e resist?ncia em determinadas popula??es. Mais recentemente, o diagn?stico molecular pela detec??o de fragmentos de DNA espec?ficos desses agentes vem sendo implementado em situa??es onde o cultivo bacteriano n?o ? vi?vel ou ? dificultado por condi??es bi?ticas ou abi?ticas. Al?m da detec??o de agentes espec?ficos, o conhecimento da diversidade bacteriana presente em alguns ambientes pode ser um importante crit?rio, como, por exemplo, para avaliar a qualidade do solo. A forma mais comum de avaliar a diversidade bacteriana se d? atrav?s do cultivo. Por?m, a maior parte das popula??es bacterianas presentes no solo n?o s?o cultiv?veis, com isso, a utiliza??o de ferramentas moleculares s?o cada vez mais utilizadas nesse tipo de an?lise. O presente estudo teve como objetivo padronizar a utiliza??o de t?cnicas independentes de cultivo para atender demandas de tr?s setores da agricultura. O primeiro cap?tulo padronizou a utiliza??o da t?cnica de DGGE (Eletroforese em Gel de Gradiente Desnaturante) para an?lise da diversidade bacteriana do leite de tetos mast?ticos e sadios de um mesmo animal. Os resultados apontaram para diferen?as no perfil bacteriano dos dois grupos analisados. O segundo cap?tulo avaliou o impacto sobre a microbiota bacteriana em solos tratados com associa??es de cascalho de perfura??o e tortas de mamona e crambe como adubos agr?colas atrav?s da an?lise de DGGE. A aplica??o da t?cnica de DGGE foi eficiente na avalia??o e confirmou que os tratamentos contribu?ram para o aumento da diversidade bacteriana nas amostras de solos estudadas. O terceiro experimento comparou a sensibilidade da t?cnica de cultivo, oficial para diagn?stico da Cria P?trida Americana (CPA), uma doen?a ap?cola causada por Paenibacillus larvae, com t?cnica de independente de cultivo por PCR do material extra?do direto do mel. Este estudo constatou que a sensibilidade da t?cnica proposta foi superior ? obtida pela t?cnica de cultivo de P. larvae. Com isso, conclui-se que as t?cnicas independentes de cultivo foram ?teis no atendimento das demandas das quest?es dos setores agr?colas contemplados no estudo.

The causative agent of the most devastating honeybee disease, American foulbrood (AFB), is the spore-forming bacterium Paenibacillus larvae. To prevent AFB outbreaks beekeepers prophylactically treat their hives with antibiotics even though it decreases the overall health of uninfected hives. A new treatment for AFB is needed due to recent legislation against using antibiotics, antibiotic resistance developing in P. larvae, and the resilience of P. larvae spores. Bacteriophages, or phages, are an attractive alternative to traditional antibiotics because of their specificity and ability to evolve alongside their target bacterium. In this study, two phage cocktails were developed for the treatment of AFB. The first cocktail was comprised of Brevibacillus laterosporus phages. B. laterosporus is a commensal microbe in most honeybee guts. When treated with B. laterosporus phages, B. laterosporus is induced to produce an antimicrobial toxin to which P. larvae is highly sensitive. Treating AFB infected hives with B. laterosporus phages was able to clear active infections at a rate of 75% as opposed to untreated hives that did not recover. However, B. laterosporus phages did not clear latent P. larvae spores and recovered hives relapsed after treatment. The second cocktail was comprised of P. larvae phages and hives treated with the second cocktail recovered at a rate of 100%, protected 100% of at-risk hives, and treated hives did not relapse with AFB suggesting neutralization of P. larvae spores. A P. larvae phage used in the second cocktail was examined to identify any spore-phage interactions. Results from modified plaque assays, fluorescence from FITC-labeled phages bound to spores, and electron microscopy images all confirm that phages bind to P. larvae spores. Phage therapy for the treatment of AFB is an exciting avenue not only as an alternative to chemical antibiotics, but rather a treatment that can neutralize P. larvae spores.

Brevibacillus laterosporus (B. laterosporus) is a pathogen difficult to distinguish from Paenibacillus larvae (P. larvae), and contributes to Colony Collapse Disorder (CCD) of honeybees. To develop a biocontrol agent to limit its presence, bacteriophages were isolated from Utah County soil samples and used to infect B. laterosporus isolated from Utah County honey and larvae samples. Since CCD is prevalent in Utah beehives, bacteriophage that infect and lyse B. laterosporus may be isolated and characterized. Pathogens were isolated from soil samples, and 16S rRNA gene tests initially identified the strains as P. larvae. Bacteriophages were isolated, purified, and amplified sufficiently to obtain images by electron microscope and genome sequencing by 454 pyrosequencing. Genomes were annotated with DNA Master, a Multiple Document Interface (MDI) program. Open reading frames (ORF's) were compared to the National Center for Biotechnology Information's (NCBI) database of primary biological sequence information via the Basic Local Alignment Search Tool (BLAST) algorithm. Later testing determined the pathogen to actually be B. laterosporus. Plaques demonstrated lytic activity, and electron microscopy revealed bacteriophages of the myoviridae family. The five sequenced genomes were composed of linear dsDNA ranging from 45,552 to 58,572 base pairs in length, 92 to 100 genes per genome, and a 38.10% to 41.44% range of G + C content. Discovering and describing new bacteriophages is a reasonably reproducible process and contributes to appreciating the diverse relationships between bacteriophage, bacteria, and eukaryota. Scientific facilitation of the bacteriophages role in limiting detrimental bacteria may contribute as an adjunctive therapy for CCD.

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Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2017-03-30T11:24:54Z No. of bitstreams: 1 texto completo.pdf: 234148 bytes, checksum: e87c51395b549c59f32533fea0cc9040 (MD5) / Made available in DSpace on 2017-03-30T11:24:54Z (GMT). No. of bitstreams: 1 texto completo.pdf: 234148 bytes, checksum: e87c51395b549c59f32533fea0cc9040 (MD5) Previous issue date: 2004-07-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Este trabalho teve como objetivo investigar a presença de Paenibacillus larvae subsp. larvae em mel de colméias do Estado do Piauí, pelo método analítico oficial brasileiro, e avaliar modificações nesta metodologia para reduzir seu limite de detecção. Esporos de P. larvae subsp. larvae não foram detectados em mel das colméias de diferentes regiões do Estado do Piauí. Comparando meios de cultura, o ágar P. larvae isolou maior número esporos desta bactéria presentes em mel e inibiu do mesmo modo contaminantes aeróbios presentes. A diluição de uma parte de mel em quatro partes de solução salina fosfatada tamponada recuperou maior número de esporos P. larvae subsp. larvae presentes em mel. As forças centrífugas 6000 g e 8000 por 30 ou 40 minutos não diferiram entre si, contudo recuperaram maior número de esporos de P. larvae subsp. larvae em comparação com a oficialmente recomendada. A redução no limite de detecção é relevante para analise de mel, principalmente, quando o patógeno encontra-se em quantidades mínimas, tanto na colméia quanto no produto comercializado. / This work had the following aims: detect the presence of Paenibacillus larvae subsp. larvae spores in honey collected in Piauí State apiaries using official Brazilian analytical method and to verify modifications in this methodology to reduce its detection limit. Spores of P. larvae subsp. larvae were not found in honey of hives from different regions of Piauí State. Comparing mediums, P. larvae agar isolated bigger spores quantities of these bacteria found in honey and inhibited, in the same way, other aerobical bacteria presented. Addition of one part of honey in four parts of phosphate buffer saline pH 7.2 isolated a bigger number of P. larvae subsp. larvae spores presented in honey. Comparisons between centrifuge forces of 6000 g and 8000 during 30 or 40 minutes showed that they were not different, however they recuperated a bigger number of P. larvae subsp. larvae spores than centrifuge force recommended in Brazilian law. Reduction in detection limit is important to honey analyses, mainly when pathogen is present in minimal quantities in hives or in commercialized product.

In diploma thesis I dealt with American foulbrood infection. At first I was focusing on disease hazards of foulbrood and I was investigating the pathogenesis of the disease bacteria Paenibacillus larvae. Then I carried out analysis of slumgum by culture test in the laboratory. I watched the bacteria P. larvae under a microscope. Next, I followed outbreaks announced by Regional Veterinary Administration. I investigated procedures of the Regional Veterinary Administration in accordance with legislation and decree during detecting the disease of AFB on the habitats. At the end, I developed a list of proclaimed outbreaks in South Bohemia into graphs and chart for the period from 2006 to 2013. The only treatment against this disease so far is burning hives, including all equipment of apiary. That is why I pointed out the importance of reporting suspected outbreaks of American foulbrood, respecting the rules and precautions by beekeepers, and beekeepers should also breed only healthy and strong beehives. Only then the spread of American foulbrood can be prevented and the originator of American foulbrood, the P. larvae, which is very durable, can be suppressed.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2016-10-11T21:19:41Z No. of bitstreams: 1 2015 - Bruno Oliveira de Carvalho.pdf: 1725115 bytes, checksum: 78e6b6f127705b066a8a0f75bbb08e88 (MD5) / Made available in DSpace on 2016-10-11T21:19:41Z (GMT). No. of bitstreams: 1 2015 - Bruno Oliveira de Carvalho.pdf: 1725115 bytes, checksum: 78e6b6f127705b066a8a0f75bbb08e88 (MD5) Previous issue date: 2015-03-17 / CAPES / The knowledge of bacterial genetic information has enable advances in many areas allowing detecting microorganisms and evaluating the production of virulence factors and resistance in certain populations. This knowledge allows the detection of microorganisms, as well as the evaluation of virulence and resistance factors production in certain populations. More recently, molecular diagnosis based on the detection of specific fragments from these agents has been implemented in no viable bacterial cultivation environments or hampered by biotic or abiotic conditions. Besides specific agent detection, bacterial diversity analysis might be an important criterion, such as assessing soil quality. Cultivation has been the most common way for evaluating bacterial diversity. However, most of soil bacteria are not cultivable; therefore, the employment of molecular tools has been increasingly used on this sort of analysis. This survey aimed standardizing the use of cultivation independent techniques for granting three agriculture sectors demands. The first chapter standardized the employment of DGGE technique for analyzing milk bacterial diversity from mastitis and healthy teats belonging to the same animal. Results pointed to differences on bacterial profile from both groups. The second chapter evaluated the impact of the employment of drill cuttings with castor beans and cramble pies association as agricultural fertilizers by DGGE analysis. The application of this technique was efficient for evaluating treatments, as well as, confirming the treatments contribution for increasing bacterial diversity in soil samples. The third chapter compared the cultivation technique sensitivity for American Foul Brood (AFB) diagnosis, a bee disease caused by Paenibacillus larvae, to cultivation independent technique by PCR. This survey demonstrated that sensitivity of the technique was greater than that one by P. larvae cultivation technique. Thus, the present survey concluded that cultivation independent techniques were useful for granting agriculture sectors demands. / O conhecimento das informa??es gen?ticas bacterianas tem permitido avan?os em diversas ?reas do conhecimento. Esse conhecimento permite detectar microrganismos, bem como avaliar a produ??o de fatores de virul?ncia e resist?ncia em determinadas popula??es. Mais recentemente, o diagn?stico molecular pela detec??o de fragmentos de DNA espec?ficos desses agentes vem sendo implementado em situa??es onde o cultivo bacteriano n?o ? vi?vel ou ? dificultado por condi??es bi?ticas ou abi?ticas. Al?m da detec??o de agentes espec?ficos, o conhecimento da diversidade bacteriana presente em alguns ambientes pode ser um importante crit?rio, como, por exemplo, para avaliar a qualidade do solo. A forma mais comum de avaliar a diversidade bacteriana se d? atrav?s do cultivo. Por?m, a maior parte das popula??es bacterianas presentes no solo n?o s?o cultiv?veis, com isso, a utiliza??o de ferramentas moleculares s?o cada vez mais utilizadas nesse tipo de an?lise. O presente estudo teve como objetivo padronizar a utiliza??o de t?cnicas independentes de cultivo para atender demandas de tr?s setores da agricultura. O primeiro cap?tulo padronizou a utiliza??o da t?cnica de DGGE (Eletroforese em Gel de Gradiente Desnaturante) para an?lise da diversidade bacteriana do leite de tetos mast?ticos e sadios de um mesmo animal. Os resultados apontaram para diferen?as no perfil bacteriano dos dois grupos analisados. O segundo cap?tulo avaliou o impacto sobre a microbiota bacteriana em solos tratados com associa??es de cascalho de perfura??o e tortas de mamona e crambe como adubos agr?colas atrav?s da an?lise de DGGE. A aplica??o da t?cnica de DGGE foi eficiente na avalia??o e confirmou que os tratamentos contribu?ram para o aumento da diversidade bacteriana nas amostras de solos estudadas. O terceiro experimento comparou a sensibilidade da t?cnica de cultivo, oficial para diagn?stico da Cria P?trida Americana (CPA), uma doen?a ap?cola causada por Paenibacillus larvae, com t?cnica de independente de cultivo por PCR do material extra?do direto do mel. Este estudo constatou que a sensibilidade da t?cnica proposta foi superior ? obtida pela t?cnica de cultivo de P. larvae. Com isso, conclui-se que as t?cnicas independentes de cultivo foram ?teis no atendimento das demandas das quest?es dos setores agr?colas contemplados no estudo.

Paenibacillus larvae is a Gram-positive sporulating bacterium that causes American foulbrood (AFB). It is one of the most dangerous bacterial pathogens of the honeybee (Apis mellifera). P. larvae spores are highly infectious to bee larvae and resist physicochemical influences. P. larvae is subtyped using repPCR with ERIC primers (Enterobacterial Repetitive Integrance Consensus) into five genotypes (ERIC I-V), which possess different colony morphology, metabolism and especially virulence. There is a significant genetic variability among isolates of P. larvae, which may contribute to differences in virulence. P. larvae isolates used in this work were obtained from clinical cases of American foulbrood as well as from a debris collected from bee hives with no American foulbrood symptoms from all over the Czech Republic in cooperation with the Beekeeping Research Institute, s.r.o., Dol. The isolates were obtained from larvae and hive debris. Both virulet and avirulet strains were sequenced using the SMRT (single molecule real time) method on the Sequel platform (PacBio). This method is suitable for Whole Genome Sequencing (WGS), because it allows sequencing of long reads with high accuracy, eliminating the effect of a large number of repetitive sequences during the genome assembly. Furthermore,...

The Paenibacillaceae family of bacteria includes two species known to infect the hives of honeybees, Paenibacillus larvae and Brevibacillus laterosporus. P. larvae, the causative agent of American Foulbrood (AFB) causes a lethal infection of honeybee larvae, while B. laterosporus is a secondary invader following European Foulbrood (EFB) infection. Increasing antibiotic resistance of P. larvae bacteria has prompted a search for alternative treatment methods for this disease. Bacteriophages are the most diverse life forms on earth and can provide important insights about the bacterial hosts they infect. However, few Paenibacillaceae phages have been isolated or characterized. In this study, the first B. laterosporus phages are characterized with respect to host range, structural morphology, and sequence similarity. The isolation and characterization of many P. larvae field isolates together with 38 novel P. larvae phages made possible the first broad phage typing study of P. larvae. Phage typing data indicated that P. larvae strains tested could be categorized into one of two groups. Comparative genomics of bacteriophages was made easier by modifying Phamerator to make it broadly accessible and usable to phage researchers throughout the world. Additionally, raw sequencing data can now be used to identify phage DNA packaging strategies that are indicative of a phage’s physical ends. Using these data, phage genomes can be published in an orientation and complementarity that reflects the physical structure of the phage chromosome, providing order and consistency that will benefit all future phage researchers.

This diploma thesis deals with a serious honey bee (Apis mellifera) disease, the American foulbrood (AFB), and with possibilities of its control. The thesis is divided into two parts, the theoretical and the experimental. The theoretical part is written as a research and contains two big chapters. The first one describes American foulbrood disease and its causative agent, bacteria Paenibacillus larvae. The second part deals with the wax moth (Galleria mellonella). The experimental part then combines above mentioned themes in an effort to find the relationship between the wax moth caterpillars and bacteria P. larvae that could possibly help to control the AFB. The aim of this work was to determine whether the wax moth can disrupt resistant layers of P. larvae spores thanks to its well adapted digestive tract and whether it could change their germination.