Nile tilapia, Oreochromis niloticus, is one of the most important farmed fish globally. One of the most serious bacterial diseases constraining global tilapia production is Francisellosis caused by Francisella noatunensis subsp. orientalis (Fno). Although outbreaks of Fno are increasing worldwide, there are no licenced commercial vaccines to prevent the disease for use on tilapia farms. Thus, the current treatment of choice is the use of antibiotics combined with increasing water temperature up to 30°C. Studies investigating the diversity of circulating Fno isolates and the immune response of tilapia elicited by vaccination against piscine francisellosis are lacking. In addition, the current conventional and molecular tools used for detection of Fno have many drawbacks, making detection of Fno a challenging process. In this study, five clinical isolates of Fno from diverse geographical locations (UK, Costa Rica, Mexico, Japan and Austria), previously characterised by morphology, genotype, antimicrobial susceptibility and virulence, were used in a proteomic study. The whole proteomic cell profile of the five isolates were homogenous by one-dimension sodium dodecyl polyacrylamide gel electrophoresis (1D-SDS-PAGE), while minor differences in the intensity of 15 proteins between the strains were observed by two-dimension SDS-PAGE (2DE), including some important virulence related proteins. The UK isolate was the most significantly different isolate when compared to the other Fno isolates in the current study. The Fno UK isolate had significantly higher abundance of 10/15 of the significantly expressed proteins including four of the essential pathogenicity and virulence related proteins (IglC, GroEL, DnaK, ClpB) compared to the other used Fno isolates. The antigenic profiles of the five Fno isolates were studied by 1D western blotting using tilapia hyper immune sera which recognised an immunodominant band of a molecular weight of ~ 17-28 kDa in all tested Fno isolates. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI/MS/MS) identified 47 proteins in this antigenic band. Some of the identified proteins are associated with Fno pathogenicity. 2D western blot analysis of the vaccine isolate (Fno UK) revealed differential antigen recognition between sera from vaccinated and non-vaccinated fish following experimental challenge (26 antigenic spots recognised by sera from vaccinated fish; 31 antigenic spots recognised by sera from vaccinated and challenged fish and 30 antigenic spots recognised by non-vaccinated and challenged fish). The identity of these proteins was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and some of them are known Francisella virulence related proteins. Bioinformatics analyses revealed diverse categories of proteins with high biological functions, however the vast majority of these proteins are involved in energy production and metabolic pathways of the bacteria. This detailed analysis will facilitate the development of cross-strain protective subunit Fno vaccines and antigen-targeted Fno diagnostics. The outer membrane proteins (OMPs) of the same five Fno isolates were extracted using the ionic detergent sarkosyl. The OMP fraction of the different isolates were separated via 1D-SDS PAGE and the digested peptides of the UK isolate were analysed by LC/ESI/MS/MS. High degree of similarity was observed in the OMP profile of the five Fno isolates with an abundant protein band at 17-28 kDa, which was found to be antigenic by 1D western blot using convalescent tilapia sera. LC/ESI/MS/MS analysis of the OMPs of the Fno UK isolate identified 239 proteins, including 44 proteins in the antigenic band (17-28 kDa). Comparison between the proteins identified in the immunogenic band of whole cell lysate and OMP fraction of the Fno UK isolate showed 30 common proteins between the two preparations, 17 proteins were identified only in the whole cell extract and 14 were identified only in OMP fraction. Outer membrane proteins (e.g. Omp-A), virulence related proteins such (e.g. IglC) and other stress related proteins (e.g. AhpC/TSA family peroxiredoxin) were more abundant in the OMP fraction than the whole cell lysate. In silico analysis enabled prediction of the function and location of the OMPs identified by Mass-spectrometry. The findings of this study provide preliminary data on bacterial surface proteins that exist in direct contact with the host immune defence during infection and offering an insight into their potential role as novel targets for Fno diagnostics and vaccine development. The efficacy of an injectable whole cell oil-adjuvanted vaccine was evaluated against challenge with heterologous Fno isolates in Nile tilapia, Oreochromis niloticus. Three duplicate groups of 130 healthy Nile tilapia (~15 g) were intraperitoneally (i.p.) injected with the vaccine, adjuvant-alone or PBS followed by an i.p. challenge with three Fno isolates from geographically distinct locations. The vaccine provided significant protection to all immunised tilapia groups with a significantly higher relative percent survival (RPS) of 82.3% against homologous challenge, compared to 69.8% and 65.9% after heterologous challenge. Protection correlated with significantly elevated specific antibody responses and western blot analysis demonstrated cross-isolate antigenicity with sera from fish post-vaccination and post-challenge. Moreover, a significantly lower bacterial burden was detected by quantitative real-time polymerase chain reaction (qPCR) in conjunction with significantly greater expression of IgM, IL-1β, TNF-a and MHCII 72 hours post-vaccination (hpv) in spleen samples from vaccinated tilapia compared to those of adjuvant-alone and control fish. The latter results suggested stimulation of protective immune responses following vaccination. In addition, a whole cell formalin killed autogenous immersion vaccine against Fno was developed using the same isolate used for the injectable vaccine. Duplicate tanks of 35 tilapia fry were immersed in the vaccine or in sterile Modified Muller Hinton broth (MMHB) diluted in tank water (1:10 dilution) for 30 s and at 30 days post-vaccination (dpv), all fish groups were immersion challenged with the homologous Fno isolate and monitored for 21 days. A moderate RPS of 43.7% was provided by the vaccine. Serum IgM levels were below the threshold in 30 % of the vaccinated fry 30 dpv. Also, the IgM levels of the vaccinated fry were not significantly different from control fry 21 days-post challenge. A recombinase polymerase amplification (RPA) assay was developed and validated for rapid detection of Fno. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in fish farms, but also by screening 78 Nile tilapia and 5 water samples collected from UK and Thailand. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other species of bacteria tested. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The Fno-RPA was rapid, giving results in approximately 6 min in contrast to the qPCR that required approximately 90 min to reach the same detection limits. Moreover, the RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to control the infection through prompt on-site detection of Fno. The overall results of this study indicated that Fno isolates from different origins share a high degree of homology in their proteomic and antigenic profile. Proteomic characterisation data of Fno isolates has contributed to understanding the diversity of Fno isolates and assisted in identifying suitable candidates for developing an effective Fno vaccine. / Moreover, this study has proven the efficacy of a cross protective Fno injection vaccine in tilapia fingerlings, with further optimisation needed for immersion vaccination of fry, and given insights into the immune response of tilapia to vaccination against francisellosis. In addition, it provided a rapid, sensitive, specific and robust molecular tool for detection of Fno that can assist surveillance and control of piscine francisellosis on tilapia farms.

O objetivo desta dissertação é avaliar a utilização dos recursos do Fundo Constitucional de Financiamento do Norte, no Estado de Roraima, no período de 2004 a 2008. Foram analisados os recursos disponibilizados para a região e a efetividade do seu emprego, ante as potencialidades sinalizadas para os 15 municípios que integram a geopolítica de Roraima. Investigou-se o porquê de um Estado que tanto se ressente de recursos para investir em atividades capazes de promover seu desenvolvimento, abre mão de valores significativos, como os que lhe tem sido colocados à disposição, ao longo dos 5 anos abrangidos pela pesquisa. As informações que lastrearam o estudo foram obtidas em várias fontes. Os subsídios específicos para a avaliação da utilização dos recursos do Fundo Constitucional de Financiamento do Norte, conseguiu-se junto ao Banco da Amazônia, em Belém, tanto em sua biblioteca, através de livros e relatórios consultados, como na Gerência de Suporte Operacional – GESOP, através de planilhas consolidando dados sobre os financiamentos contratados, no período sob análise, a nível de setor, atividades e de municípios beneficiados em todo o Estado. O suporte teórico do trabalho foi enriquecido pelas idéias sobre o círculo vicioso da pobreza, a teoria do desenvolvimento desequilibrado, a teoria dos pólos de crescimento, a teoria da base de exportação e a teoria da base econômica, esta última, dando embasamento ao estudo, por se mostrar adequada à sua utilização, considerando-se que regiões subdesenvolvidas, além da variável exportação, conta também com outras variáveis exógenas como capital de empréstimo e risco, pagamento de fatos e transferências governamentais. Concluiu-se que o Estado de Roraima não vem utilizando o volume integral dos recursos do FNO. Apenas, 25% dos recursos disponibilizados, ou seja, R$ 163 milhões, dos R$ 666 milhões alocados para consumo pelos produtores e empresários foram efetivamente aplicados. Os municípios do Estado, todos com potencial para o desenvolvimento da agricultura e pecuária, pouco utilizaram os recursos do FNO. Verificouse que dos recursos do FNO que ficaram em Roraima, o Município de Boa Vista absorveu 37%, o Município de Bonfim com 16%, os Municípios de Alto Alegre e Iracema, com 14% cada e os Caracaraí, Cantá e Mucajaí, com 13% cada um, do volume total dos recursos desembolsados no período. O Município de Pacaraima não teve um único financiamento contratado e o de Uiramutã, inserido na Reserva Raposa Serra do Sol foi beneficiado, ao longo dos 5 anos da pesquisa, com financiamentos que atingiram apenas o valor global de R$ 88 mil reais, o que é inexpressivo. As causas que contribuíram para que as metas esperadas pelo Banco da Amazônia, com relação a Roraima, não fossem atingidas, estão diretamente relacionadas às seguintes ocorrências: ausência da presença física do Banco nos municípios (87% das unidades do Estado não possuem sequer uma agência do Banco); à questão fundiária não equacionada, dificultando a celebração de contratos com pessoas que ainda não detém títulos definitivos de suas terras; a centralização do poder decisório das operações, fora do Estado; a carência de bons projetos; o excesso de burocracia na apreciação dos pleitos; a demora, para atendimento dos pedidos de empréstimos e financiamentos e à falta de capacidade de gerenciamento, iniciativa e de conhecimento dos potenciais demandantes de crédito. / The objective of this dissertation is to evaluate the use of resources for the Constitutional Fund for North Financing in the State of Roraima, from 2004 to 2008. We analyzed the available resources for the region and the effectiveness of their usage, before the appointed potential of the 15 municipalities which compose Roraima geopolitics. We investigated why a state that resents so much of wherewithal to invest in activities with capacity to promote its development, relinquishes significant amounts, such as those that have been made available over the five years covered by this survey. The informations that ballasted this study were obtained from various sources. The specific aids to evaluate the use of the wherewithal from the Constitutional Fund for the North Financing were obtained from the Bank of Amazonia, in Belém, both in its library, through books and consulted reports, as from the operational support management – GESOP through worksheets consolidating data regarding the contracted funding during the period under review, by sector level, activities and benefited municipalities all over the State. The theoretical support of this work was enriched by the ideas about the vicious circle of poverty, the theory of unequal development, the theory of poles of economic growth, the export base theory and the economic basis theory, this one giving basement to the study due to being appropriated for utilization, considering that the underdeveloped regions, besides the variable export, also count on other exogenous variables such as loans and risk capital, payment of facts and government transfers. The conclusion was that the state of Roraima hasn’t been using the total volume of the wherewithal from FNO. Only 25% of the available amount, that is, R$ 163 million from the R$ 666 million allocated for use by producers and entrepreneurs were effectively applied. The state municipalities, all with potential for developing the agriculture and livestock, used a small part of the resources from FON. It was found that from the resources of FNO who remained in Roraima, the city of Boa Vista had absorbed 37%, the City of Bonfim 16%, the cities of Alto Alegre and Iracema, with 14% each and Caracaraí, Cantá and Mucajaí, with 13% each, from all the total disbursed funds during the period. The city of Pacaraima had no contracted financing and Uiramutã, in Raposa Serra do Sol reservation, received, over the five years researched, financings that reached just the total amount of R$ 88 thousand Reais, which is unimpressive. The causes that contributed for the non-fulfillment of the goals expected by the Bank of Amazonia for Roraima are directly related to the following events: lack of physical presence of the Bank in the cities (there aren’t bank agencies in 87% of the cities), the unsolved land issue, making it difficult to conclude contracts with people who still hasn’t got the definitive titles of their lands; centralization of the power to decide the operations outside the state; the lack of good projects; excess of bureaucracy in the projects appraisement; the delay in attending the loans and financing applications and the lack of management capacity, initiative and knowledge of potential credit applicants.

O objetivo desta dissertação é avaliar a utilização dos recursos do Fundo Constitucional de Financiamento do Norte, no Estado de Roraima, no período de 2004 a 2008. Foram analisados os recursos disponibilizados para a região e a efetividade do seu emprego, ante as potencialidades sinalizadas para os 15 municípios que integram a geopolítica de Roraima. Investigou-se o porquê de um Estado que tanto se ressente de recursos para investir em atividades capazes de promover seu desenvolvimento, abre mão de valores significativos, como os que lhe tem sido colocados à disposição, ao longo dos 5 anos abrangidos pela pesquisa. As informações que lastrearam o estudo foram obtidas em várias fontes. Os subsídios específicos para a avaliação da utilização dos recursos do Fundo Constitucional de Financiamento do Norte, conseguiu-se junto ao Banco da Amazônia, em Belém, tanto em sua biblioteca, através de livros e relatórios consultados, como na Gerência de Suporte Operacional – GESOP, através de planilhas consolidando dados sobre os financiamentos contratados, no período sob análise, a nível de setor, atividades e de municípios beneficiados em todo o Estado. O suporte teórico do trabalho foi enriquecido pelas idéias sobre o círculo vicioso da pobreza, a teoria do desenvolvimento desequilibrado, a teoria dos pólos de crescimento, a teoria da base de exportação e a teoria da base econômica, esta última, dando embasamento ao estudo, por se mostrar adequada à sua utilização, considerando-se que regiões subdesenvolvidas, além da variável exportação, conta também com outras variáveis exógenas como capital de empréstimo e risco, pagamento de fatos e transferências governamentais. Concluiu-se que o Estado de Roraima não vem utilizando o volume integral dos recursos do FNO. Apenas, 25% dos recursos disponibilizados, ou seja, R$ 163 milhões, dos R$ 666 milhões alocados para consumo pelos produtores e empresários foram efetivamente aplicados. Os municípios do Estado, todos com potencial para o desenvolvimento da agricultura e pecuária, pouco utilizaram os recursos do FNO. Verificouse que dos recursos do FNO que ficaram em Roraima, o Município de Boa Vista absorveu 37%, o Município de Bonfim com 16%, os Municípios de Alto Alegre e Iracema, com 14% cada e os Caracaraí, Cantá e Mucajaí, com 13% cada um, do volume total dos recursos desembolsados no período. O Município de Pacaraima não teve um único financiamento contratado e o de Uiramutã, inserido na Reserva Raposa Serra do Sol foi beneficiado, ao longo dos 5 anos da pesquisa, com financiamentos que atingiram apenas o valor global de R$ 88 mil reais, o que é inexpressivo. As causas que contribuíram para que as metas esperadas pelo Banco da Amazônia, com relação a Roraima, não fossem atingidas, estão diretamente relacionadas às seguintes ocorrências: ausência da presença física do Banco nos municípios (87% das unidades do Estado não possuem sequer uma agência do Banco); à questão fundiária não equacionada, dificultando a celebração de contratos com pessoas que ainda não detém títulos definitivos de suas terras; a centralização do poder decisório das operações, fora do Estado; a carência de bons projetos; o excesso de burocracia na apreciação dos pleitos; a demora, para atendimento dos pedidos de empréstimos e financiamentos e à falta de capacidade de gerenciamento, iniciativa e de conhecimento dos potenciais demandantes de crédito. / The objective of this dissertation is to evaluate the use of resources for the Constitutional Fund for North Financing in the State of Roraima, from 2004 to 2008. We analyzed the available resources for the region and the effectiveness of their usage, before the appointed potential of the 15 municipalities which compose Roraima geopolitics. We investigated why a state that resents so much of wherewithal to invest in activities with capacity to promote its development, relinquishes significant amounts, such as those that have been made available over the five years covered by this survey. The informations that ballasted this study were obtained from various sources. The specific aids to evaluate the use of the wherewithal from the Constitutional Fund for the North Financing were obtained from the Bank of Amazonia, in Belém, both in its library, through books and consulted reports, as from the operational support management – GESOP through worksheets consolidating data regarding the contracted funding during the period under review, by sector level, activities and benefited municipalities all over the State. The theoretical support of this work was enriched by the ideas about the vicious circle of poverty, the theory of unequal development, the theory of poles of economic growth, the export base theory and the economic basis theory, this one giving basement to the study due to being appropriated for utilization, considering that the underdeveloped regions, besides the variable export, also count on other exogenous variables such as loans and risk capital, payment of facts and government transfers. The conclusion was that the state of Roraima hasn’t been using the total volume of the wherewithal from FNO. Only 25% of the available amount, that is, R$ 163 million from the R$ 666 million allocated for use by producers and entrepreneurs were effectively applied. The state municipalities, all with potential for developing the agriculture and livestock, used a small part of the resources from FON. It was found that from the resources of FNO who remained in Roraima, the city of Boa Vista had absorbed 37%, the City of Bonfim 16%, the cities of Alto Alegre and Iracema, with 14% each and Caracaraí, Cantá and Mucajaí, with 13% each, from all the total disbursed funds during the period. The city of Pacaraima had no contracted financing and Uiramutã, in Raposa Serra do Sol reservation, received, over the five years researched, financings that reached just the total amount of R$ 88 thousand Reais, which is unimpressive. The causes that contributed for the non-fulfillment of the goals expected by the Bank of Amazonia for Roraima are directly related to the following events: lack of physical presence of the Bank in the cities (there aren’t bank agencies in 87% of the cities), the unsolved land issue, making it difficult to conclude contracts with people who still hasn’t got the definitive titles of their lands; centralization of the power to decide the operations outside the state; the lack of good projects; excess of bureaucracy in the projects appraisement; the delay in attending the loans and financing applications and the lack of management capacity, initiative and knowledge of potential credit applicants.

O objetivo desta dissertação é avaliar a utilização dos recursos do Fundo Constitucional de Financiamento do Norte, no Estado de Roraima, no período de 2004 a 2008. Foram analisados os recursos disponibilizados para a região e a efetividade do seu emprego, ante as potencialidades sinalizadas para os 15 municípios que integram a geopolítica de Roraima. Investigou-se o porquê de um Estado que tanto se ressente de recursos para investir em atividades capazes de promover seu desenvolvimento, abre mão de valores significativos, como os que lhe tem sido colocados à disposição, ao longo dos 5 anos abrangidos pela pesquisa. As informações que lastrearam o estudo foram obtidas em várias fontes. Os subsídios específicos para a avaliação da utilização dos recursos do Fundo Constitucional de Financiamento do Norte, conseguiu-se junto ao Banco da Amazônia, em Belém, tanto em sua biblioteca, através de livros e relatórios consultados, como na Gerência de Suporte Operacional – GESOP, através de planilhas consolidando dados sobre os financiamentos contratados, no período sob análise, a nível de setor, atividades e de municípios beneficiados em todo o Estado. O suporte teórico do trabalho foi enriquecido pelas idéias sobre o círculo vicioso da pobreza, a teoria do desenvolvimento desequilibrado, a teoria dos pólos de crescimento, a teoria da base de exportação e a teoria da base econômica, esta última, dando embasamento ao estudo, por se mostrar adequada à sua utilização, considerando-se que regiões subdesenvolvidas, além da variável exportação, conta também com outras variáveis exógenas como capital de empréstimo e risco, pagamento de fatos e transferências governamentais. Concluiu-se que o Estado de Roraima não vem utilizando o volume integral dos recursos do FNO. Apenas, 25% dos recursos disponibilizados, ou seja, R$ 163 milhões, dos R$ 666 milhões alocados para consumo pelos produtores e empresários foram efetivamente aplicados. Os municípios do Estado, todos com potencial para o desenvolvimento da agricultura e pecuária, pouco utilizaram os recursos do FNO. Verificouse que dos recursos do FNO que ficaram em Roraima, o Município de Boa Vista absorveu 37%, o Município de Bonfim com 16%, os Municípios de Alto Alegre e Iracema, com 14% cada e os Caracaraí, Cantá e Mucajaí, com 13% cada um, do volume total dos recursos desembolsados no período. O Município de Pacaraima não teve um único financiamento contratado e o de Uiramutã, inserido na Reserva Raposa Serra do Sol foi beneficiado, ao longo dos 5 anos da pesquisa, com financiamentos que atingiram apenas o valor global de R$ 88 mil reais, o que é inexpressivo. As causas que contribuíram para que as metas esperadas pelo Banco da Amazônia, com relação a Roraima, não fossem atingidas, estão diretamente relacionadas às seguintes ocorrências: ausência da presença física do Banco nos municípios (87% das unidades do Estado não possuem sequer uma agência do Banco); à questão fundiária não equacionada, dificultando a celebração de contratos com pessoas que ainda não detém títulos definitivos de suas terras; a centralização do poder decisório das operações, fora do Estado; a carência de bons projetos; o excesso de burocracia na apreciação dos pleitos; a demora, para atendimento dos pedidos de empréstimos e financiamentos e à falta de capacidade de gerenciamento, iniciativa e de conhecimento dos potenciais demandantes de crédito. / The objective of this dissertation is to evaluate the use of resources for the Constitutional Fund for North Financing in the State of Roraima, from 2004 to 2008. We analyzed the available resources for the region and the effectiveness of their usage, before the appointed potential of the 15 municipalities which compose Roraima geopolitics. We investigated why a state that resents so much of wherewithal to invest in activities with capacity to promote its development, relinquishes significant amounts, such as those that have been made available over the five years covered by this survey. The informations that ballasted this study were obtained from various sources. The specific aids to evaluate the use of the wherewithal from the Constitutional Fund for the North Financing were obtained from the Bank of Amazonia, in Belém, both in its library, through books and consulted reports, as from the operational support management – GESOP through worksheets consolidating data regarding the contracted funding during the period under review, by sector level, activities and benefited municipalities all over the State. The theoretical support of this work was enriched by the ideas about the vicious circle of poverty, the theory of unequal development, the theory of poles of economic growth, the export base theory and the economic basis theory, this one giving basement to the study due to being appropriated for utilization, considering that the underdeveloped regions, besides the variable export, also count on other exogenous variables such as loans and risk capital, payment of facts and government transfers. The conclusion was that the state of Roraima hasn’t been using the total volume of the wherewithal from FNO. Only 25% of the available amount, that is, R$ 163 million from the R$ 666 million allocated for use by producers and entrepreneurs were effectively applied. The state municipalities, all with potential for developing the agriculture and livestock, used a small part of the resources from FON. It was found that from the resources of FNO who remained in Roraima, the city of Boa Vista had absorbed 37%, the City of Bonfim 16%, the cities of Alto Alegre and Iracema, with 14% each and Caracaraí, Cantá and Mucajaí, with 13% each, from all the total disbursed funds during the period. The city of Pacaraima had no contracted financing and Uiramutã, in Raposa Serra do Sol reservation, received, over the five years researched, financings that reached just the total amount of R$ 88 thousand Reais, which is unimpressive. The causes that contributed for the non-fulfillment of the goals expected by the Bank of Amazonia for Roraima are directly related to the following events: lack of physical presence of the Bank in the cities (there aren’t bank agencies in 87% of the cities), the unsolved land issue, making it difficult to conclude contracts with people who still hasn’t got the definitive titles of their lands; centralization of the power to decide the operations outside the state; the lack of good projects; excess of bureaucracy in the projects appraisement; the delay in attending the loans and financing applications and the lack of management capacity, initiative and knowledge of potential credit applicants.