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The dramatic feeding-related activities of the Chagas' disease vector, Rhodnius prolixus are under neurohormonal regulation of serotonin and various neuropeptides. One such family of neuropeptides, the insect kinins, possesses diuretic, digestive and myotropic activities in many insects. In R. prolixus, they co-localize with the corticotropin-releasing factor (CRF)-like diuretic hormone (DH) in neurosecretory cell bodies and their abdominal neurohaemal sites. Additionally, kinins are present in endocrine cells of the midgut and are known to stimulate hindgut and midgut contractions. Through the experimentation presented in this dissertation, the cloning and spatial expression of the R. prolixus kinin (Rhopr-kinin) transcript is described. Physiological bioassays demonstrate the myostimulatory effects of selected Rhopr-kinin peptides and also illustrate the augmented responses of hindgut contractions to co-application of Rhopr-kinin and Rhopr-CRF/DH. The irreversible effects of two synthetic kinin analogs on the hindgut relative to the native kinins also exhibit the prospective biotechnological significance of this study.

The dramatic feeding-related activities of the Chagas' disease vector, Rhodnius prolixus are under neurohormonal regulation of serotonin and various neuropeptides. One such family of neuropeptides, the insect kinins, possesses diuretic, digestive and myotropic activities in many insects. In R. prolixus, they co-localize with the corticotropin-releasing factor (CRF)-like diuretic hormone (DH) in neurosecretory cell bodies and their abdominal neurohaemal sites. Additionally, kinins are present in endocrine cells of the midgut and are known to stimulate hindgut and midgut contractions. Through the experimentation presented in this dissertation, the cloning and spatial expression of the R. prolixus kinin (Rhopr-kinin) transcript is described. Physiological bioassays demonstrate the myostimulatory effects of selected Rhopr-kinin peptides and also illustrate the augmented responses of hindgut contractions to co-application of Rhopr-kinin and Rhopr-CRF/DH. The irreversible effects of two synthetic kinin analogs on the hindgut relative to the native kinins also exhibit the prospective biotechnological significance of this study.

Made available in DSpace on 2017-07-10T14:17:04Z (GMT). No. of bitstreams: 1 Mayaha.pdf: 4416602 bytes, checksum: 8b663982eaff70076dc570733618b841 (MD5) Previous issue date: 2013-08-01 / Bombyx mori multiple nucleopolyedrovirus (BmMNPV) is an entomopathogenic virus of the Baculoviridaefamily, genera Alphabaculovirus, which infects the silkworm (Bombyx Mori) and causes nuclear polyhedrosis disease. This virus is poliorganotrophic and a series of tissues are known as targets; however, literature does not present information in regards to the pylorus, segment of the hindgut, present in the transition with the midgut and responsible for regulating the passage of food towards the ileum. The organ is, thus, of fundamental importance in the ending of the digestive process, affecting the insect s metabolic balance and, the present study sought to analyze its susceptibility and cytopathology in regards to BmMNPV, geographic isolate of Paraná, Brazil. Moreover, due to the complexity of this segment, its general morphology will also be analyzed. In order to do so, hybrid B.mori caterpillars at 5º instar were inoculated experimentally with a viral suspension of BmMNPV. On different day post-inoculation (dpi), from 2º to 9ºdpi, segments of the intestine, containing the pylorus, with its subdivisions (posterior interstitial ring, pyloric cone and pyloric valve), were dissected, following the routine histological processing for paraffin wax blockage and dyes in haematoxylin and eosin, for general morphology analysis, and in modified Azan staining, for cytopathology. Segments also processed to form scanning electronic microscope for analized of morphological details. So the pylorus general morphology the caterpillars of B. mori, was similar to that observed in other insects. The cytopathology showed that the anterior area of the posterior interstitial ring is a secondary target of infection for the BmMNPV, after 5º dpi, and its infectious cycle is similar to that described in literature. In the hypertrophic nucleus one there the formation of viroplasm, place for production of enveloped nucleocapsids or virions, following the formation and development of the polyhedra. Later on, cytolysis occurred, promoting the liberation of polyhedra and the spreading of the disease, mainly in the B. mori creation sheds. The posterior area of the posterior interstitial ring, the pyloric cone and the pyloric valve did not reveal any traces of infection by BmMNPV and, thus, resistance mechanisms involving genetic factors could be acting. The knowledge obtained from this work will contribute in the establishment of the infectious cycle of this important entomopathogenic virus, which may lead to an epizootic that can negatively affect the whole productive chain of silk / Bombyx mori multiple nucleopolyedrovirus (BmMNPV) é um vírus entomopatogênico da família Baculoviridae, gênero Alphabaculovirus, que infecta o bicho-da-seda (Bombyx mori) e causa a doença poliedrose nuclear. Este vírus é poliorganotrófico e uma série de tecidos são conhecidos como alvos; contudo, a literatura não apresenta informações sobre o piloro, segmento do intestino posterior, presente na transição com o médio e responsável pela regulação da passagem do alimento em direção ao ileo. O órgão é assim de fundamental importância na finalização do processo digestório, afetando o equilíbrio metabólico do inseto e, o presente estudo objetivou analisar sua susceptibilidade e citopatologia frente ao BmMNPV, isolado geográfico do Paraná, Brasil. Ainda, devido a complexidade deste segmento, também será analisada sua morfologia geral. Para tanto, lagartas híbridas de B. mori no 5° instar foram inoculadas experimentalmente com uma suspensão viral de BmMNPV. Em diferentes dias pós-inoculação (dpi), do 2° ao 9° dpi, segmentos do intestino, contendo o piloro, com suas subdivisões (anel intersticial posterior, cone pilórico e válvula pilórica), foram dissecados, seguindo o processamento histológico de rotina para emblocamento em parafina e colorações em hematoxina e eosina, na análise da morfologia geral, e em Azan modificado, para a citopatologia. Segmentos também foram processados em microscopia eletrônica de varredura para análise de detalhes morfológicos. Assim, a morfologia geral do piloro das lagartas da B. mori foi semelhante à observada em outros insetos. A citopatologia mostrou que a área anterior do anel intersticial posterior é alvo secundário de infecção pelo BmMNPV, a partir do 5° dpi, e seu ciclo infeccioso é semelhante ao descrito em literatura. No núcleo hipertrófico houve a formação do viroplasma, local de produção dos nucleocapsídeos envelopados ou virions, seguindo a formação e o desenvolvimento dos poliedros. Posteriormente, ocorreu a citólise, promovendo a liberação dos poliedros e disseminação da doença, principalmente nos barracões de criação de B. mori. A área posterior do anel intersticial posterior, o cone pilórico e a válvula pilórica não revelaram quaisquer indícios de infecção pelo BmMNPV e, desta forma, mecanismos de resistência envolvendo fatores genéticos poderiam estar atuando. Os conhecimentos obtidos irão contribuir no estabelecimento do ciclo infeccioso deste importante vírus entomopatogênico, cuja conseqüência de uma epizootia pode afetar negativamente toda a cadeia produtiva da seda

L’équilibre de l’écosystème microbien du caecum (Ca) et du colon ventral droit (Cn) équin peut être rompu par un changement brusque d’aliments et par une ration riche en amidon, et conduire à l’apparition de coliques. Chez des chevaux soumis à ces deux pratiques alimentaires, nous avons étudié les modifications de ces écosystèmes, de l’écosystème microbien fécal et de paramètres sanguins. Nous avons également testé la supplémentation composée de Saccharomyces cerevisiae et de microalgues Schizochytrium limacinum dans le cadre d’une collaboration avec Alltech. Nous avons montré que les écosystèmes microbiens du Ca et du Cn, stables lors d’un régime foin, pouvaient être rapidement modifiés par le stress provoqué par les deux pratiques alimentaires. Chez les chevaux supplémentés certains taxons bactériens potentiellement fibrolytiques ont augmenté dans le Ca et le Cn, et les dysbioses résultant d’un régime riche en amidon ont été limitées. Nous avons identifié plusieurs paramètres fécaux et sanguins reflétant les variations de l’écosystème microbien du Ca et Cn lors d’un changement de régime: les groupes bactériens fonctionnels, les abondances relatives de taxons bactériens (la famille XIII de Clostridiales, Succinivibrionaceae et des genres appartenant à Ruminococcaceae, Lachnospiraceae ou Prevotellaceae), les proportions et le ratio d’AGV, la concentration de valerate, les lipopolysaccharides fécaux ainsi que le beta-hydroxybutyrate sanguin. Ces paramètres semblent prometteurs pour diagnostiquer les dysbioses du gros intestin équin et prévenir l’apparition des coliques chez le cheval. / The balance of the microbial ecosystem of the caecum (Ca) and the right ventral colon (Cn) of the horse can be disturbed by an abrupt change of feed and by a high-starch diet, and lead to appearance of colic. We investigated the alterations of these ecosystems, of the faecal microbial ecosystem and of blood parameters in horses subjected to these two dietary practices. In addition, we tested a supplementation combining the yeast Saccharomyces cerevisiae and the microalgae Schizochytrium limacinum, in a context of partnership with Alltech. We showed that the Ca and Cn microbial ecosystems were stable under a hay diet, and can rapidly be modified under a stress caused by the two dietary practices. In supplemented horses, some potential fibrolytic bacterial taxa increased in the Ca and Cn, and the dysbiosis resulting from a high-starch diet were limited. We identified several faecal and blood parameters reflecting the variations of the Ca and Cn microbial ecosystem under a change of diet: the bacterial functional groups, the relative abundances of bacterial taxa (family XIII of Clostridiales, Succinivibrionaceae and genera belonging to Ruminococcaceae, Lachnospiraceae or Prevotellaceae), the proportion of acetate and propionate, the VFA ratio, the valerate concentration, the faecal lipopolysaccharides and the blood beta-hydroxybutyrate. These parameters appeared promising to diagnose dysbiosis in the proximal hindgut and thus prevent colic appearance in horses.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Escherichia coli O157 is a major foodborne pathogen. The bovine gut is the primary reservoir and the organism is shed in the feces, which serves as the major source of contamination. The objectives of our research on E. coli O157 were to 1) determine prevalence and characterize presence in the rectoanal mucosa of cattle, 2) rationalize preferential prevalence in the hindgut, 3) evaluate fecal prevalence and concentration in relation to carcass contamination 4) determine the efficacy of preharvest intervention strategies to reduce fecal prevalence and/or concentration in cattle. We determined that E. coli O157 isolated from the rectoanal mucosa were similar to fecal isolates. We evaluated methods of enumeration in cattle feces to identify fecal samples with > 10[superscript3] and 10[superscript4] CFU of E. coli O157/g. We observed that prevalence of E. coli O157 on carcasses was correlated with high-shedders. We identified gluconic acid as a substrate which stimulates growth of E. coli O157 in fermentations with ruminal microbial or fecal microbial inocula from cattle. This may explain the preference to persist or colonize the hindgut because gluconic acid is a component of colonic mucin. Additionally, we evaluated the effects of cattle diets (two grain types and two grain processing methods), which present different amounts of fermentable starch to the hindgut, on prevalence of E. coli O157. Dry-rolled grain-based diets reduced prevalence of E. coli O157, possibly because of increased flow of starch to the hindgut. Competitive exclusion cultures of E. coli in in vitro fermentations reduced E. coli O157 in ruminal microbial inoculum, but in fecal microbial inoculum cultures were only efficacious when gluconic acid also was added. Lastly, we evaluated a vaccine which targeted the siderophore receptor/porin proteins of E. coli O157. The vaccine reduced prevalence, the total number of days cattle tested positive, and the total number of days cattle were identified as high-shedders of E. coli O157. In summary, our research adds further knowledge to the literature about E. coli O157 in the hindgut, provides methods to identify high-shedding animals, demonstrates the importance of high-shedding animals, and offers information about potential preharvest interventions.

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