Orientador: Ana Valéria Colnaghi Simionato Cantú / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T23:35:40Z (GMT). No. of bitstreams: 1 Orlando_EduardoAdilson_M.pdf: 785459 bytes, checksum: 50e5f02a98813d34ba94c694183e9030 (MD5) Previous issue date: 2013 / Resumo: A classe de antibióticos tetraciclinas é amplamente empregada na aquicultura e possui especial dificuldade analítica devido a uma estrutura complexa e grande interação com os componentes da matriz cárnea. Neste trabalho foram avaliadas diferentes técnicas de extração de resíduos de tetraciclinas em filés de tilápias. Analisou-se oxitetraciclina, doxiciclina, tetraciclina e clortetraciclina por HPLC-fluorescência nas condições: gradiente de fase móvel, com fase orgânica composta por MeOH:ACN (1:1, v/v) e fase aquosa pH 7,00 contendo Na2COOH (37,5 mmol L), CaCl2 (17,5 mmol L) e EDTA (12,5 mmol L) e detecção em 385/528 nm (lexc/lem). Foram avaliados os métodos de extração: partição líquido-líquido; extração em fase sólida utilizando as fases estacionárias fenil, C18 e polimérica Oasis-HLB; concentração em resina XAD 16; e QuEChERS; que foram otimizados utilizando planejamento experimental fatorial fracionário e comparados quanto à eficiência de extração. A partição líquido-líquido e o protocolo QuEChERS apresentaram baixas eficiências de extração dos analitos (14-30%). O emprego da resina polimérica Amberlite XAD 16 apresentou boa eficiência (40-60%), mas com baixa precisão e grande consumo de tempo. O emprego de SPE apresentou os melhores resultados, com destaque para a fase fenil (58-76%), sendo então este método validado com relação as figuras de mérito seletividade, linearidade, exatidão, precisão, limite de detecção e quantificação, mostrando-se adequado para a investigação dos analitos em níveis de resíduos em filés de tilápia. Um total de 26 amostras comerciais foram analisadas, sendo que apenas uma delas apresentou resíduo de 42,0 ± 8,4 ng g de Oxitetraciclina, estando abaixo do limite máximo de resíduo no estabelecido no Brasil de 200 ng g / Abstract: The indiscriminate use of antibiotics in aquaculture can generate residues in meat intended for human consumption and cause the arising of bacterial resistance. The tetracycline antibiotics class is widely used and has ultimate analytical difficulties due to a complex structure and large interaction with components of meat matrix. In this work different techniques were evaluated for extraction of tetracycline residues in tilapia fillets. Oxytetracycline, doxycycline, tetracycline and chlortetracycline were analyzed by HPLCfluorescence under the following conditions: mobile phase gradient; organic phase composed by MeOH:ACN (1:1, v/v) and aqueous phase containing Na2COOH (37,5 mmol L), CaCl2 (17,5 mmol L) e EDTA (12,5 mmol L) at pH 7.00 and detection at 385/528 nm (lexc/lem). The evaluated extraction methods were: liquid-liquid partition; solid phase extraction (SPE) using stationary phases phenyl, C18 and Oasis HLB; concentration on adsorbent resin XAD 16; and QuEChERS. The methods were optimized using fractional factorial experimental design and compared in terms of extraction efficiency. The liquidliquid partition and QuEChERS protocol showed low extraction efficiencies (14-30%). The use of polymeric resin Amberlite XAD 16 showed a good efficiency (40-60%) but with low precision and large time consumption. The use of SPE showed the best results and was validated according to the merit figures selectivity, linearity, accuracy, precision, limit of detection and quantification, making it adequate for the investigation of analytes at levels of residues in tilapia fillets. A total of 26 real samples were analyzed and only one sample exhibited Oxytetracycline residue of 42,0 ± 8,4 ng g, being below the maximum residue limit established in Brazil of 200 ng g / Mestrado / Quimica Analitica / Mestre em Química

Orientadores: Felix Guillermo Reyes Reyes, Jonas Augusto Rizzato Paschoal / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-20T20:20:58Z (GMT). No. of bitstreams: 1 PilcoQuesada_Silvia_M.pdf: 1450390 bytes, checksum: a021e048252c2afe9ae8f4892c08fc56 (MD5) Previous issue date: 2012 / Resumo: A aquicultura é um setor de produção de alimentos que apresenta grande crescimento em todo o mundo. O Brasil possui um alto potencial de expansão da atividade aquícola, com produção promissora de espécies exóticas como a tilápia (Oreochromis niloticus) e nativas como o pacu (Piaractus mesapotamicus) e o tambaqui (Colossoma macropomum),. Como em todo sistema intensivo de produção, o uso de medicamentos veterinários na produção aquícola se faz necessário para o controle e tratamento de doenças, visando garantir os rendimentos de produção esperados. Essas doenças podem ser causadas por parasitas, bactérias, fungos e vírus. Dentre os medicamentos mais utilizados estão os antimicrobianos, uma vez que as bacterioses são indicadas como um dos fatores principais de limitação da produção aquícola no mundo todo. Dentre os antimicrobianos comumente utilizados na aquicultura mundial destaca-se o grupo das fluoroquinolonas (FQ). O risco potencial da presença de resíduos de antimicrobianos nos produtos aquícolas é um assunto preocupante. Dentre os fatores de preocupação estão o uso irresponsável e não controlado, a falta de respeito às boas práticas veterinárias e carência de alternativas de medicamentos veterinários específicos para uso na aquicultura. Como consequência, tem-se a preocupação com a questão da resistência microbiana, assunto este muito discutido mundialmente, e que aflige e impacta tanto o próprio sistema de produção aquícola, como a saúde humana e o meio ambiente. Quando os resíduos dos antimicrobianos estão acima dos valores de limites máximos de resíduos (LMR) estabelecidos, passam a representar risco potencial à saúde do consumidor. Deste modo, o desenvolvimento de métodos analíticos para determinar os resíduos de fármacos veterinários presentes em produtos de origem animal se faz necessário. Assim, o objetivo deste trabalho foi desenvolver e validar um método para a determinação simultânea de resíduos de norfloxacina (NOR), danofloxacina (DAN), enrofloxacina (ENR) e ciprofloxacina (CIP) em filés de tilápia (Oreochromis niloticus) e de pacu (Piaractus mesopotamicus), utilizando a cromatografia líquida acoplada à espectrometria de massas sequencial (LC-MS/MS QToF). Para a validação analítica foram considerados os seguintes parâmetros: linearidade, sensibilidade, seletividade, efeito matriz, precisão intra-dia (com amostras fortificadas e amostras incorridas) e inter-dias, exatidão, limite de detecção, limite de quantificação, limite de decisão e capacidade de detecção. Os resultados obtidos a partir da validação apresentaram conformidade com as recomendações dos guias de validação da Comunidade Europeia e do Ministério da Agricultura, Pecuária e Abastecimento, indicando que o método é apropriado para a determinação de FQ em filé de peixe. Nenhuma das amostras (n=31) adquiridas do comércio e analisadas pelo método validado apresentou resíduos de DAN, NOR, ENR e CIP em níveis detectáveis / Abstract: Aquaculture is a food production sector that presents worldwide growth. Aquaculture activity in Brazil exhibits a high potential for expansion, that includes promising production of exotic as tilapia (Oreochromis niloticus) and native species pacu (Piaractus mesapotamicus) and tambaqui (Colossoma macropomum). As in every intensive production system, the use of veterinary drugs in aquaculture production is necessary for control and treatment of diseases, in order to guarantee the expected productivity. Those diseases can be caused by parasites, bacteria, fungi and viruses. Antibiotics are among the most widely used drugs, since bacterial diseases are listed as one of the main factors limiting the worldwide aquaculture production. The fluoroquinolones (FQ) antibiotic family is the most commonly used in aquaculture. The risk of the presence of antibiotics residues in aquaculture products is a matter of concern, the irresponsible and uncontrolled use of antibiotics, the disrespect to good veterinary practices, and the lack of alternatives of veterinary specific drugs for aquaculture use, are some of the matters of concern. As a consequence, it arises a current issue related to microbial resistance which may affect different areas such as aquaculture system, human health and the environment. Potential risk to the consumer health is presented when the antimicrobial residues in foods are above the established maximum residue limits (MRL). Therefore, it becomes necessary the development and validation of analytical methods in order to determine the veterinary drugs residues present in foods of animal origin. Thus, the objective of this work was to develop and validate a method for the simultaneous determination of norfloxacin (NOR), danofloxacin (DAN), enrofloxacin (ENR) and ciprofloxacin (CIP) residues in tilapia (Oreochromis niloticus) and pacu (Piaractus mesopotamicus) fillets, using liquid chromatography associated to mass spectrometry (LC-MS/MS QToF). To conduct the validation of the method the following parameters were considered: linearity, sensitivity, selectivity, matrix effect, intra-day (using spiked and incurred samples) and inter-day precision, accuracy, detection limit, quantification limit, decision limit and detection capability. The obtained results are in conformity with the guidelines recommendations from the European Community, and the Ministry of Agriculture, Livestock and Supply, demonstrating that the method is suitable for the determination of FQ in fish fillet. None of the samples (n=31) acquired and analyzed by the validated method presented residues of DAN, NOR, ENR and CIP at detectable levels / Mestrado / Ciência de Alimentos / Mestra em Ciência de Alimentos

La production de tilapias et surtout celle du tilapia du Nil Oreochromis niloticus (Linnaeus, 1758) connaît depuis 30 ans, une impressionnante croissance liée à ses nombreux atouts pour l'aquaculture. Toutefois, le contrôle du sexe conditionne la rentabilité de cette filière. En effet chez le tilapia du Nil le mâle présente une meilleure croissance que la femelle. Depuis les années 70, la production de populations monosexes mâles de tilapia du Nil est pratiquée dans la quasi-totalité des fermes aquacoles afin de bénéficier de la meilleure croissance des mâles par rapport aux femelles et de contrôler la reproduction précoce et continue chez cette espèce. Actuellement, l'inversion hormonale du sexe par la 17α-methyltestosterone (17MT) constitue la technique la plus utilisée pour produire les cohortes monosexes mâles. Toutefois, l'utilisation des hormones pour la production de poissons marchands soulève de nombreuses questions relevant de la sécurité alimentaire comme de la protection des travailleurs piscicoles et de l'environnement. D'autres approches sont donc recherchées. Elles reposent sur des approches génétiques, ou sur les effets masculinisants des fortes températures ou enfin sur l'utilisation d'anti-aromatase. Dans cette thèse, nous avons comparé les effets de trois traitements, utilisant respectivement la 17MT, le fadrozole (un inhibiteur de l'aromatase) et les fortes températures, sur des caractères d'intérêt zootechniques (survie, croissance) et sur l'expression de gènes clés de la différenciation du sexe dans les gonades et dans le cerveau des mâles induits (mâles 17MT, mâles anti-aromatase et thermo-néomâles). Parallèlement les mêmes analyses ont été menées sur des mâles et des femelles génétiques. Les fortes températures produisent des mâles dont la croissance est similaire à celle des mâles génétiques, confortant l'idée que les traitements thermiques pourraient constituer une alternative écologique aux utilisations des hormones pour la production de populations monosexes mâles chez le tilapia, sous réserve de sélectionner des souches à forte thermosensibilité. Le rôle positif des androgènes et/ou l'effet inhibiteur des œstrogènes sur la croissance est discuté. L'analyse des profils d'expression de gènes clés de la différenciation sexuelle suggère des régulations différentes dans le cerveau et dans les gonades. En particulier, l'amh et cyp19a1b sont très fortement stimulées dans le cerveau des mâles traités par la température ; inversement l'amh est inhibée dans le cerveau des individus traités par le fadrozole, suggérant une régulation directe ou indirecte de l'amh par l'aromatase. Dans les gonades des mâles induits, les niveaux de dmrt1 et amh sont surexprimés par rapport à ce qui est observé chez les mâles génétiques. Ce travail mériterait d'être complété par a) des études sur la fertilité des mâles induits b) des dosages des taux circulants de stéroïdes et de protéines dans le plasma c) des analyses histologiques ou immunohistochimiques du cerveau et des gonades et d) l'étude de l'expression d'autres gènes.Mots-clés : Tilapia du Nil / Masculinisation / Température / Hormones / Croissance / Gènes / Due to the genuine advantages of this species (fast growth, spontaneous breeding in captivity), the aquaculture production of tilapias, and especially of the Nile tilapia Oreochromis niloticus (L.), has been growing steadily over the past 30 years. Nevertheless, the success of this production is largely dependent on the sex control efficiency, in order to take advantage of the better growth of males and to avoid uncontrolled breeding that can cause crowding and dwarfism. The use of male monosex populations has thus become a standard in tilapia aquaculture since the 1970s. The dietary administration of 17α-methyltestosterone (17MT) is still the major masculinising procedure used in tilapia farming nowadays. However, it raises issues concerning the environment, fish farmers and consumers'health. More recently, alternatives to hormonal sex reversal have been evaluated, i.e. the use of YY males, treatment using aromatase inhibitors or elevated temperatures during the early life stages. In the present study, the effects of fadrozole (an aromatase inhibitor), elevated temperatures and 17MT have been compared on the survival and growth of XX fish, and as regards to the expression patterns in the brain and gonads of key genes implicated in fish sex differentiation. Comparisons seen in these induced-males were performed with genetic XX females and XY males. The growth rates of fish raised at elevated temperature were similar to those of genetic males, thereby emphasizing the relevance of this method as an alternative to hormonal sex reversal of Nile tilapia, when using highly thermosensitive strains. Whether the growth of tilapia is stimulated by androgens or depressed by estrogens is discussed. The analysis of expression profiles of the genes involved in sexual differentiation reveals different patterns in the brain and in the gonads. Amh and cyp19a1b are strongly stimulated in the brain of XX neomales treated by elevated temperatures. Conversely, amh is inhibited in the brain of fish treated with fadrozole, suggesting that amh is directed or indirectly regulated by aromatase. In the gonads of XX neomales, the levels of dmrt1 and amh are much higher than in genetic XY males. Further studies should analyse a) the fertility of neomales, b) the plasmatic levels of steroids and proteins, c) the histology and immunohistochemistry of the brain and the gonads, and d) the expression of other genes.Keywords : Nile tilapia / Masculinisation / Temperature / Hormones / Growth / Genes

Caracteriza molecularmente las bacterias patógenas causantes de enfermedades en cultivo intensivo cerrado de tilapia Oreochromis niloticus con sistema Biofloc de una piscigranja del departamento de Lima durante los años 2013, 2014 y 2015. Se aislaron 28 cepas bacterianas de órganos internos, siendo identificadas bioquímicamente por técnicas habituales y el uso de sistemas miniaturizados API 20 E y NE. Las pruebas de sensibilidad antimicrobiana se realizaron empleando el método de Kirby-Bauer. La confirmación diagnóstica se determinó por la Reacción en Cadena de Polimerasa (PCR) y secuenciamiento del gen 16S ARNr. Se logró identificar y caracterizar molecularmente las cepas bacterianas siguientes: Edwardsiella tarda en número de seis (6), Citrobacter freundii (2), Aeromonas caviae (5), V. cholerae (1), Vibrio alginolyticus (1), Plesiomonas shigelloides (5), Shewanella algae (5) y S. putrefaciens (1). Se determinó el perfil filogenético de ocho especies bacterianas, las cuales se encuentran en su respectivo clado. El 100% de las cepas de E. tarda, V. alginolyticus y V. cholerae fueron sensibles a la totalidad de antibióticos evaluados. Algunas bacterias presentaron resistencia a oxitetraciclina y ácido nalidíxico como C. freundii (50%), P. shigelloides (80 y 60%), S. putrefaciens (100%) y A. caviae (40 y 60%). Los signos externos frecuentemente observados en peces de las fases de crecimiento y comercial fueron: eritema en aletas pectorales, vientre y alrededor del ano, siendo provocados por E. tarda, C. freundii, P. shigelloides, V. alginolyticus, V. cholerae, S. putrefaciens, S. algae y A. caviae. Entre los signos internos se presentó el hígado congestivo cuyos agentes causales fueron V. alginolyticus, V. cholerae, S. putrefaciens y A. caviae El estudio histopatológico reveló necrosis caseosa en el ventrículo cardiaco, hígado, bazo, riñón posterior y gónadas de peces afectados por E. tarda. En ejemplares sintomáticos, de los cuales se aislaron P. shigelloides y A. caviae se observaron lesiones en el tracto gastrointestinal. Asimismo, en individuos afectados por S. putrefaciens, S. algae y V. algynoliticus se apreció congestión de capilares sinusoides y necrosis de hepatocitos. Existe una gran diversidad de géneros bacterianos que afectan a las tilapias bajo sistema biofloc durante la fase productiva de crecimiento y comercial. / Tesis

Orientador: Margarida Maria Barros / Coorientador: Filipe Giardini Pereira Bonfim / Resumo: Os Peixes foram alimentados com dietas contendo níveis de pó de gengibre (GgP) para verificar seus efeitos sobre o desempenho produtivo e resistência à infecção bacteriana. Um grupo de 540 tilápias-do-Nilo (8,2 g ± 0,34) foi distribuído aleatoriamente em 36 aquários de 250 L (15 peixes / tanque) e alimentadas com seis dietas práticas, uma sem a adição de gengibre (0 GgP, dieta basal, controle positivo) e níveis crescentes de suplementação de 0,25% (0,25 GgP), 0,5% (0,5 GgP), 0,75% (0,75 GgP), 1% (1 GgP) e 1,5% da dieta GgP (1,5 GgP). Estas dietas foram formuladas para conter 29% de proteína digestível e 18 MJ de energia digestível kg-1. Após 30 dias de alimentação, determinou-se o desempenho produtivo, os parâmetros hematológico e imunológico e a atividade das enzimas do sistema antioxidantes. Em seguida, os peixes foram submetidos a desafio com Aeromonas hydrophila e a mortalidade foi registrada por 15 dias. Os mesmos parâmetros e atividade de enzimas do sistema antioxidantes foram determinados após esse tempo. Peixes alimentados com dietas contendo níveis acima de 0,5 GgP apresentaram maior peso final e taxa de crescimento específica. Maior ingestão de ração foi determinada nos peixes alimentados com dietas contendo 1GgP e 1,5 GgP (P <0,05). Quanto aos parâmetros hematológicos e imunológicos, antes ou após o desafio bacteriano, peixes alimentados com 0 GgP e 1,5 GgP apresentaram os piores resultados para todos os parâmetros. Os peixes alimentados com 1 GgP mostraram a maior... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Not available / Mestre

Caracteriza molecularmente las bacterias patógenas causantes de enfermedades en cultivo intensivo cerrado de tilapia Oreochromis niloticus con sistema Biofloc de una piscigranja del departamento de Lima durante los años 2013, 2014 y 2015. Se aislaron 28 cepas bacterianas de órganos internos, siendo identificadas bioquímicamente por técnicas habituales y el uso de sistemas miniaturizados API 20 E y NE. Las pruebas de sensibilidad antimicrobiana se realizaron empleando el método de Kirby-Bauer. La confirmación diagnóstica se determinó por la Reacción en Cadena de Polimerasa (PCR) y secuenciamiento del gen 16S ARNr. Se logró identificar y caracterizar molecularmente las cepas bacterianas siguientes: Edwardsiella tarda en número de seis (6), Citrobacter freundii (2), Aeromonas caviae (5), V. cholerae (1), Vibrio alginolyticus (1), Plesiomonas shigelloides (5), Shewanella algae (5) y S. putrefaciens (1). Se determinó el perfil filogenético de ocho especies bacterianas, las cuales se encuentran en su respectivo clado. El 100% de las cepas de E. tarda, V. alginolyticus y V. cholerae fueron sensibles a la totalidad de antibióticos evaluados. Algunas bacterias presentaron resistencia a oxitetraciclina y ácido nalidíxico como C. freundii (50%), P. shigelloides (80 y 60%), S. putrefaciens (100%) y A. caviae (40 y 60%). Los signos externos frecuentemente observados en peces de las fases de crecimiento y comercial fueron: eritema en aletas pectorales, vientre y alrededor del ano, siendo provocados por E. tarda, C. freundii, P. shigelloides, V. alginolyticus, V. cholerae, S. putrefaciens, S. algae y A. caviae. Entre los signos internos se presentó el hígado congestivo cuyos agentes causales fueron V. alginolyticus, V. cholerae, S. putrefaciens y A. caviae El estudio histopatológico reveló necrosis caseosa en el ventrículo cardiaco, hígado, bazo, riñón posterior y gónadas de peces afectados por E. tarda. En ejemplares sintomáticos, de los cuales se aislaron P. shigelloides y A. caviae se observaron lesiones en el tracto gastrointestinal. Asimismo, en individuos afectados por S. putrefaciens, S. algae y V. algynoliticus se apreció congestión de capilares sinusoides y necrosis de hepatocitos. Existe una gran diversidad de géneros bacterianos que afectan a las tilapias bajo sistema biofloc durante la fase productiva de crecimiento y comercial. / Tesis

Piscine francisellosis in an infectious emerging bacterial disease that affects several marine and fresh water fish species worldwide, including farmed salmon, wild and farmed cod, farmed tilapia and several ornamental species, for which no commercial treatment or vaccine exists. During 2011 and the first semester of 2012, chronic episodes of moderate to high levels of mortality with nonspecific clinical signs, and widespread multifocal white nodules as the most consistent gross pathological lesion were experienced by farmed tilapia fingerlings at two different locations in Northern Europe. In this study such outbreaks of granulomatous disease were diagnosed as francisellosis with a genus-specific PCR, and 10 new isolates of the bacterium including the one named STIR-GUS-F2f7, were recovered on a new selective “cysteine blood-tilapia” agar and cysteine heart agar with bovine haemoglobin. Ultrastructural observations of the pathogen in Nile tilapia (O. niloticus) tissues suggested the secretion of outer membrane vesicles (OMVs) by the bacterial cells during infection in these fish. This represented the first documented report of isolation of pathogenic Francisella strains from tilapia in Europe. The phenotypic characterisation indicated that isolates recovered were able to metabolise dextrin, N-acetyl-D glucosamine, D-fructose, α-D-glucose, D-mannose, methyl pyruvate, acetic acid, α-keto butyric acid, L-alaninamide, L-alanine, L-alanylglycine, L-asparagine, L-glutamic acid, L-proline, L-serine, L-threonine, inosine, uridine, glycerol, D L-α-glycerol phosphate, glucose-1-phosphate and glucose-6-phosphate. The predominant structural fatty acids of the isolates were 24:1 (20.3%), 18:1n-9 (16.9%), 24:0 (13.1%) 14:0 (10.9%), 22:0 (7.8%), 16:0 (7.6%) and 18:0 (5.5%). Anti-microbial resistance analyses indicated that STIR-GUS-F2f7 was susceptible to neomycin, novobiocin, amikacin, ciprofloxacin, imipenem, gatifloxacin, meropenem, tobramycin, nitrofurantoin, and levofloxacin using the quantitative broth micro-dilution method, while the qualitative disc diffusion method indicated susceptibility to enrofloxacin, kanamycin, gentamicin, tetracycline, oxytetracycline, florfenicol, oxolinic acid and streptomycin. The use of the following housekeeping genes: mdh, dnaA, mutS, 16SrRNA-ITS-23SrRNA, prfB putA rpoA, rpoB and tpiA indicated 100% similarity with other isolates belonging to the subspecies F. noatunensis orientalis (Fno). Koch’s postulates were successfully fulfilled by establishing an intraperitoneal injection (IP) challenge model with STIR-GUS-F2f7 in Nile tilapia. Moreover, the challenge model was used to investigate the susceptibility of 3 genetic groups of tilapia to STIR-GUS-F2f7. The lowest amount of bacteria required to cause mortality was 12 CFU/ml and this was seen as early as only 24 hours post infection in the red Nile tilapia and in the wild type after 26 days, no mortalities were seen in the species O. mossambicus with this dose. The mortality in red O. niloticus was significantly higher than that of the other two tilapia groups when 12 and 120 CFU/fish were injected. It was also observed that when a dose of 1200 CFU/ml was used, the mortality in O. niloticus wild type was significantly lower than that of the other two tilapia groups and no differences were seen among the 3 groups when the highest dose (1.2 x105 CFU/fish) was used. The median lethal dose (LD50) of O. niloticus wild type was the most stable during the experiment (values around 104 CFU/ml) and the highest of the three groups after day 25 post infection. At the end of the experiment (day 45) the LD50 was 30 CFU/ml in the red Nile tilapia, 2.3x104 CFU/ml for the wild type and 3.3x102 CFU/ml for O. mossambicus. This pattern, where the LD50 of the red tilapia was lower than that of the other two groups, was observed during the whole experiment. The outcomes of these experiments suggested that the red Nile tilapia family appeared to be the most susceptible while the wild type Nile tilapia family the most resistant. The complete genome of STIR-GUS-F2f7 was sequenced using next generation sequencing (NGS) Illumina Hi-Seq platform™, and the annotation of the assembled genome predicted 1970 protein coding sequences and 63 non-coding rRNA sequences distributed in 328 sub-systems. The taxonomy of the species Francisella noatunensis was revised using genomic-derived parameters form STIR-GUS-F2f7 and other strains in combination with a polyphasic approach that included ecologic, chemotaxonomic and phenotypic analyses. The results indicated that STIR-GUS-F2f7 and all the other strains from warm water fish represent a new bacterial species for which the name Francisella orientalis was assigned. Moreover the description of F. noatunensis was emended and the creation of a new subspecies within this taxon i.e. Francisella noatunensis subsp. chilense was proposed. The results of this study led to the development of a highly efficacious vaccine to protect tilapia against francisellosis.

Szeto Tsz Kwan Leo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 112-120). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Abbreviations --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Heavy metals contaminations in Shing Mun River --- p.1 / Chapter 1.1 --- Importance of copper regulation and role of liverin copper metabolism --- p.6 / Chapter 1.1.1 --- Role of copper --- p.6 / Chapter 1.1.2 --- Toxicity due to unbalanced copper regulation --- p.7 / Chapter 1.1.3 --- Function of liver in copper detoxification --- p.9 / Chapter 1.2 --- Aims and rationale of this research --- p.11 / Chapter Chapter 2 --- Heavy metal concentrations of tilapia samples collected from Shing Mun River --- p.12 / Chapter 2.1 --- Introduction --- p.12 / Chapter 2.1.1 --- Sampling sites - Fo Tan and Siu Lek Yuen Nullah --- p.12 / Chapter 2.1.2 --- Tilapia samples collected from the sites --- p.16 / Chapter 2.1.3 --- Tilapia as a study model --- p.18 / Chapter 2.1.4 --- Bioavailability of heavy metals in water --- p.19 / Chapter 2.1.5 --- Metal content in liver --- p.20 / Chapter 2.1.6 --- Aim of this chapter --- p.20 / Chapter 2.2 --- Materials and Methods --- p.22 / Chapter 2.2.1 --- Collection of control and field samples --- p.22 / Chapter 2.2.2 --- Heavy metal concentrations determination --- p.23 / Chapter 2.2.3 --- Homogenization of liver cells --- p.24 / Chapter 2.2.4 --- Subcellular fractionation --- p.24 / Chapter 2.2.5 --- Determination of copper and zinc content in each subcellular fraction --- p.253 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Physical data --- p.27 / Chapter 2.3.2 --- Metal concentrations in liver and muscle --- p.27 / Chapter 2.3.3 --- Copper and zinc subcellular distribution in liver cell --- p.33 / Chapter 2.4 --- Discussion --- p.36 / Chapter 2.4.1 --- Difference in metal concentration between sites --- p.36 / Chapter 2.4.2 --- Copper contamination in water and fish organ (muscle and liver) from the Shing Mun River --- p.36 / Chapter 2.4.3 --- Comparison of metal content in muscle and liver at Fo Tan site with previous studies --- p.39 / Chapter 2.4.4 --- Copper and zinc concentrations in the liver of tilapia --- p.42 / Chapter 2.4.5 --- Copper and zinc sebcellular distribution in the liver of tilapia --- p.43 / Chapter Chapter 3 --- Column chromatography of hepatic proteins from tilapias --- p.44 / Chapter 3.1 --- Transport of metals from circulatory system to liver --- p.44 / Chapter 3.1.1 --- Copper transporting plasma proteins in vertebrates --- p.44 / Chapter 3.1.2 --- Copper uptake into hepatocytes --- p.45 / Chapter 3.1.3 --- Intracellular metabolism of copper --- p.48 / Chapter 3.1.4 --- Mechanism of copper toxicity following excess accumulation --- p.49 / Chapter 3.1.5 --- Aim of this chapter --- p.50 / Chapter 3.2 --- Materials and Methods --- p.51 / Chapter 3.2.1 --- Purification of liver cytosolic proteins by gel-filtration column chromatography --- p.51 / Chapter 3.2.2 --- Copper content detection in elution --- p.52 / Chapter 3.2.3 --- Analysis of peaks from elution profile using tricine gel SDS PAGE --- p.53 / Chapter 3.3 --- Results --- p.55 / Chapter 3.3.1 --- Gel-filtration liquid chromatography elution profiles --- p.55 / Chapter 3.3.2 --- SDS PAGE analysis of peaks in elution profiles --- p.51 / Chapter 3.4 --- Discussion --- p.54 / Chapter 3.4.1 --- Comparison of gel filtration profiles of sample liver cytosol between sites and sexes --- p.64 / Chapter 3.4.2 --- Possible proteins in peaks found in the gel filtration profiles --- p.64 / Chapter 3.4.3 --- Common copper-indeced proteins --- p.67 / Chapter 3.5 --- Conclusion --- p.70 / Chapter Chapter 4 --- Two-dimensional electrophoresis of hepatic cutosol of tilapias caught from Shing Mun River and copper-treated HEPA T1 cell --- p.72 / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.1.1 --- The need of ´بin vitro' experiment --- p.72 / Chapter 4.1.2 --- Choice of cell line --- p.73 / Chapter 4.1.3 --- Aim of this chapter --- p.74 / Chapter 4.2 --- Materials and Methods --- p.76 / Chapter 4.2.1 --- HEPA T1 cell cultivation --- p.76 / Chapter 4.2.2 --- Copper exposure of HEPA T1 cell --- p.77 / Chapter 4.2.3 --- Subcellular protein extraction of the copper-treated HEPA T1 cells --- p.77 / Chapter 4.2.4 --- Bicinchoninic Acidic (BCA) Protein Assay --- p.79 / Chapter 4.2.5 --- Two-dimensional gel electrophoresis --- p.79 / Chapter 4.3 --- Results --- p.83 / Chapter 4.3.1 --- Graphical presentation of spots observed on 2-dimensional gel of field samples and copper-injected samples --- p.33 / Chapter 4.3.2 --- Graphical presentation of spots detected on 2-dimensional gel of HEPAT1 cells --- p.84 / Chapter 4.3.3 --- Comparison of matched spots on 2-dimensional gels among control and copper-treated HEPAT1 cells --- p.97 / Chapter 4.4 --- Discussion --- p.105 / Chapter 4.4.1 --- Comparison of the spot patterns between field sample and copperOtreated HEPA T1 cells --- p.105 / Chapter 4.5 --- Conclusion --- p.107 / Chapter Chapter 5 --- General Discussions --- p.108 / Chapter 5.2 --- Research Overview --- p.108 / Chapter 5.2 --- Characterization of metal binding proteins from the cytosol of liver of tilapia --- p.109 / REFERENCES --- p.112

Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005.

Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005