Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-16T19:56:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_LeonardoQuintanaSoaresLopes.pdf: 1473450 bytes, checksum: eb7d00c9d4a740c17c2d7fd0ee98c234 (MD5) / Made available in DSpace on 2018-08-16T19:56:49Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_LeonardoQuintanaSoaresLopes.pdf: 1473450 bytes, checksum: eb7d00c9d4a740c17c2d7fd0ee98c234 (MD5) Previous issue date: 2015-03-20 / The American Foulbrood Disease (AFB) is a fatal larval bee disease. The etiologic agent is the bacteria Paenibacillus larvae, being highly contagious due to the capacity of this microorganism to form spores. The treatment involves incineration of all contaminated materials, leading to high losses. The Glycerol Monolaurate (MLG) is a compound with a known antimicrobial potential, however its use is impaired due to the low solubility in water and the high melting point. In this context, the nanostructuration of some drugs offers advantages like better stability and solubility, increasing effectiveness and reducing toxic effects. The present study aimed to produce, characterize and evaluate the antimicrobial activity, the toxicity in bees and the ecotoxicity of MLG nanoparticles. The nanoparticles produced showed a diameter of 209.9 nm, polidispersion index of 0.044 and zeta potential of -23.4 mV. The study of the stability parameters showed that even after 120 days, the system was stable. The images of transmission electronic microscopy showed the nanometric size and spherical forms. The determination of inhibitory and bactericidal concentration showed that is necessary 142.8 μg/ml of the formulation (MLG nanoparticle) to obtain a bacteriostatic effect and of 285 μg/ml to obtain a bactericidal effect. The assay in bees showed that the free compound has a high toxicity while the nanoparticles showed a low toxic effect, reducing the number of dead bees. When analyzed the ecotoxicity, the springtail test soil results in high toxicity of the free compound, while the nanostructured formulation didn’t influence in mortality either in reproduction of organisms, showing that nanocapsules aren’t toxic to the ecosystem. Concluding, the developed formulation shows positive results in the action to combat AFB beyond not cause damage in bees and in soil, and can be future alternative to the treatment or prevention of AFB. / A American Foulbrood Disease (AFB) é uma doença fatal que acomete as abelhas em seu estado larval. Seu agente etiológico é a bactéria Paenibacillus larvae, sendo altamente contagiosa devido à capacidade de esse microrganismo formar esporos. O tratamento atualmente envolve a incineração de todos os materiais contaminados levando a grandes prejuízos. O monolaurato de glicerol (MLG) é um composto com um conhecido potencial antimicrobiano, entretanto, seu uso é prejudicado devido ao seu baixo grau de solubilidade em água e o seu alto ponto de fusão. Neste contexto, a nanoestruturação de fármacos oferece vantagens como aumento de estabilidade em ambientes adversos, melhora as questões de solubilidade, aumentando a eficácia e reduzindo os efeitos tóxicos. O presente estudo teve como objetivo produzir, caracterizar e avaliar a atividade antimicrobiana, toxicidade em abelhas e ecotoxicidade de nanocápsulas contendo MLG. As nanocápsulas produzidas apresentaram um diâmetro médio de 209,9 nm, índice de polidispersão 0,044 e potencial zeta de -23,4 mV. O estudo de parâmetros de estabilidade mostrou que mesmo passados 120 dias, o sistema se manteve estável. As imagens geradas por microscopia eletrônica de transmissão mostraram o tamanho nanométrico além de formas características esféricas. A determinação da concentração inibitória e bactericida mínima mostrou que são necessários 142,8 μg/ml da formulação (Nanocápsulas de MLG) para se obter um efeito bacteriostático e de 285,7 μg/ml para obtenção de um efeito bactericida. O ensaio nas abelhas mostrou que o composto livre possui uma alta toxicidade, enquanto que as nanocápsulas apresentaram um efeito pouco tóxico, reduzindo o número de abelhas mortas. Quando avaliada a ecotoxicidade, o teste no solo com colêmbolos resultou em uma alta toxicidade do composto livre, enquanto que a formulação nanoestruturada não influenciou na mortalidade nem na reprodução dos organismos, mostrando que as nanocápsulas não são tóxicas para o ecossistema. Concluindo, a formulação desenvolvida apresenta resultados positivos em sua ação no combate da AFB além de não causar dano às abelhas e ao solo, podendo ser uma alternativa futura para o tratamento ou prevenção da AFB.

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.

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Paenibacillus larvae is a gram-positive spore-forming bacterium that affects and kills the larvae of the honey bee (Apis mellifera) and causes the American foulbrood disease. Adults bees do not become infected, but they transmit tenacious spores within the hive and between hives and can infect larvae while caring for them. It is not allowed by law to treat bees in the Czech Republic, but the recommended preventive method for reducing the amount of spores in the hive is the shook swarm method (bees are moved to a new clean hive and the old hive is destroyed with all brood and supplies). The aim of this work was to detect and quantify P. larvae in bee workers using the quantitative real-time polymerase chain reaction (qPCR). In the first experiment, the two set of samples were taken - bees before and after the shook swarm method, but the expected decrease in spores in the samples taken after shook swarm was not confirmed, and conversely, non-specific products were amplified. In the second experiment, the presence of P. larvae spores in samples from heavily infected hives (with clinical symptoms of American foulbrood) and from hives with almost no findings of P. larvae spores, both originating from the same habitat, were compared. In this case, the differences were clearly visible. There were not...