Made available in DSpace on 2015-03-03T11:52:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-27Bitstream added on 2015-03-03T12:06:09Z : No. of bitstreams: 1 000805074_20160227.pdf: 82407 bytes, checksum: 27387f7880642489826cb800af053e88 (MD5) Bitstreams deleted on 2016-02-29T12:16:27Z: 000805074_20160227.pdf,. Added 1 bitstream(s) on 2016-02-29T12:17:26Z : No. of bitstreams: 1 000805074.pdf: 475905 bytes, checksum: 38da9ded440f4702eb18c90ebac18bfd (MD5) / Identificou-se no presente estudo o agente patogênico causador de surtos de estreptococose em cachara e surubins como sendo a Lactococcus garvieae. Considerando que a lactococose é uma doença emergente de importância econômica mundial, que gera surtos de mortalidade em massa em diversas espécies de peixes, avaliamos a possibilidade de prevenção e controle desta patogenia com o uso de vacinas. Após confirmação da patogenicidade da bactéria pelo Postulado de Koch, foram formuladas duas bacterinas inativadas com formalina, uma aquosa e uma oleosa, com emprego de adjuvante. Surubins híbridos foram imunizados via intraperitoneal com ambas bacterinas e com PBS. Para avaliar a eficiência das vacinas foi comparada a potência dos imunobiológicos desenvolvidos na sobrevivência dos animais, na cinética de anticorpos 15, 30 e 60 dias após a vacinação e na modulação da resposta imune após desafio bacteriano. Melhores níveis de proteção foram observados com a imunização com bacterina oleosa, onde foi observado maior nível de anticorpos circulantes e cujo valor de porcentagem relativa de sobrevivência foi de 81,7%. O presente fez o primeiro registro de surto de septicemia hemorrágica causada por Lactococcus garvieae em surubins híbridos e cacharas, e demonstrou que a imunidade dos surubins pode ser estimulada com emprego de vacinas, e que a bacterina oleosa proporciona proteção mais eficaz contra a lactococose / The present study identified the pathogen causing outbreaks of estreptococose in cachara and surubins as being the Lactococcus garvieae. Whereas lactococose as an emerging disease of worldwide economic importance, that generates outbreaks of mass mortality in several species of fish, we evaluated the possibility of prevention and control of this pathogen with the use of vaccines. After confirming the pathogenicity of the bacterium by Koch postulate, two inactivated bacterins with formalin, an aqueous and an oily, with the use of adjuvant, were formulated. Surubins hybrids were immunized intraperitoneally with PBS and both bacterins. To evaluate the efficacy of the vaccines was compared the potency of the biopharmaceuticals developed in survival of the animals, on the kinetic of antibody 15, 30 and 60 days after vaccination and in the modulation of the immune response after bacterial challenge. Best levels of protection were observed with immunization with oily bacterin, where were observed higher level of circulating antibodies and whose value relative survival percentage was 81.7 %. This work made the first recorded outbreak of haemorrhagic septicemia caused by Lactococcus garvieae in hybrid surubins and cacharas, and demonstrated that the immunity of surubins can be stimulated through the use of vaccines, and oily bacterin provides more effective protection against lactococose

Orientadora: Fabiana Pilarski / Coorientadora: Márcia Mayumi Ishikawa / Banca: Ricardo Massato Takemoto / Banca: Luiz Augusto do Amaral / Resumo: Neste trabalho foi registrada a primeira ocorrência de infecção por Citrobacter freundii, Lactococcus lactis subsp. lactis e Enterococcus faecalis associadas a surtos de doenças em bagres Sul Americanos. Além do isolamento das bactérias, também foi realizada a caracterização bioquímica e o perfil de susceptibilidade a antimicrobianos dos isolados. Para o isolamento, foram utilizados bagres provenientes de surtos em diferentes fazendas-berçário no Estado do Mato Grosso do Sul e também de criações em laboratório. Os peixes com sinais clínicos de bacteriose foram submetidos à necropsia e biópsia de rim e encéfalo para isolamento bacteriano em meios de cultura apropriados. Os isolados obtidos foram submetidos à coloração de Gram, teste da oxidase, catalase, produção de hemólise e identificação presuntiva através de kits comerciais API 20E ou API 20 Strep. A análise de susceptibilidade a 15 antimicrobianos foi realizada pelo método da difusão em disco com o Agar Muller Hinton. Foram obtidos dois isolados de C. freundii proveniente de cachara, que apresentaram multiresistência a 66,7 % dos antimicrobianos testados. Nos ensaios de infecção experimental, a doença foi reproduzida com sucesso, caracterizada como septicemia hemorrágica, com enterite acentuada e com reisolamento de C. freundii dos tecidos acometidos. Outros 57 isolados obtidos do rim e do encéfalo da cachara foram caracterizadas como cocos gram positivos associadas a meningoencefalite, identificadas como Lactococcus lactis subsp. lactis e Enterococcus faecalis. Todos os isolados apresentaram multiresistência aos antimicrobianos testados. A bactéria L. lactis subsp. lactis foi sensível somente a ampicilina e penicilina. Todas nossos isolados de E. faecalis foram sensíveis a sulfazotrim e a amoxilina e ao ácido clavulânico / Abstract: The first occurrence of infection by Citrobacter freundii, Lactococcus lactis subsp. lactis and Enterococcus faecalis associated with outbreaks of diseases in South American catfish was recorded in this study. The isolates were biochemically characterized and the profile antimicrobial susceptibility performed. For isolation, fingerlings of catfish from sequential outbreaks at different farms nursery in the state of Mato Grosso do Sul and also creations in the laboratory were used. Fingerlings with clinical signs of bacterial infection were necropsied and biopsy of the kidney and brain obtained for bacterial isolation in appropriate culture media. The isolates were subjected to Gram staining, oxidase and catalase test, hemolysis production and presumptive identification using commercial kits API 20E and API 20 Strep. The analysis of susceptibility to 15 antimicrobials was performed by the disc diffusion method with the Muller Hinton Agar. Were obtained two isolates of C. freundii from cachara, which showed multiresistance to 66.7% of the antimicrobials tested. In experimental challenge, the disease was successfully reproduced, characterized as hemorrhagic septicemia, with severe enteritis, and reisolation of C. freundii from infected tissue was obtained. Another 57 isolates obtained from kidney and brain of catfish were characterized as gram-positive cocci associated with meningoencephalitis, identified as Lactococcus lactis subsp. lactis and Enterococcus faecalis. All isolates showed multidrug resistance to antimicrobials. The bacteria L. lactis subsp. lactis was only sensitive to ampicillin and penicillin. All our isolates of E. faecalis were sensitive to sulfazotrim and amoxilin and to clavulanic acid / Mestre

Orientadora: Fabiana Pilarski / Banca: Dalton José Carneiro / Banca: Maurício Laterça Martins / Resumo: Há inúmeras razões para a exploração da aquicultura mundial, principalmente no Brasil, país apontado pela FAO, como um dos poucos capazes de produzir alimentos suficientes para atender a demanda mundial nos próximos 30 anos. A piscicultura brasileira destaca-se pela produção de tilápias em tanques-rede, e este sistema foi alavancado graças ao desenvolvimento das técnicas de incubação artificial de ovos e masculinização de pós-larvas. Todavia, a masculinização pode sobrecarregar o metabolismo dos indivíduos, uma vez que, durante a fase de pós-larva, o sistema digestório destes animais ainda é imaturo. A utilização de hormônios esteroides aliado a níveis elevados de proteína, tem como consequência alterações fisiopatológicas, como o comprometimento hepático, que poderão perdurar por todo o ciclo de produção, com isso, os peixes podem tornar-se mais susceptíveis a patógenos oportunistas ou serem submetidos a tratamentos equivocados. Este trabalho propõe a utilização da homeopatia na ausência de produtos específicos e registrados para uso na aquicultura no Brasil. O medicamento foi avaliado em duas apresentações: complexo homeopático Fator Pró-digestão Inicial ® administrado na dieta de pós-larvas de tilápias-do-Nilo e o medicamento homeopático Isoterápico, gotejado na água dos peixes (2,5 gotas/minuto-1), ambos durante o ciclo inicial de produção (incluindo o período de masculinização) até 70 dias. O estudo foi realizado em uma piscicultura comercial do estado de São Paulo sendo avaliado: desempenho produtivo, sobrevivência, prevalência parasitária e realizados exames histopatológicos do fígado e brânquias das pós-larvas. A homeopatia, nas duas apresentações, melhorou a sobrevivência dos peixes, o complexo homeopático Fator Pró-digestão Inicial® e o seu veículo (sacarose) melhoraram o desempenho produtivo em relação aos grupos controle ... / Abstract: There are innumerous reasons to explore the worldwide aquaculture. Brazil has been pointed by FAO, as one of the few countries able to produce food to handle worldwide demand for the next 30 years., The aquaculture, as farming activity, is unique, and demands a vast knowledge in limnology, ictiology and ecology. Tilapia production in net-cages made a huge advance after the discovery of artificial incubation of eggs and masculinization of fingerlings. During this phase, the digestory system of fish is still immature and with the utilization of steroid hormones and high protein diets, it can overload the system, causing physiopathological alterations that will endure all the production cycle. With that, fish could be more susceptible to opportunistic agents. This way, it is desired to use products that can make fish with a higher ability to answer the challenges of farming. As a regulation therapy, homeopathy uses the natural force of the organism to reestablish the homeostasis. One of the unique application of population homeopathy is the easier to administrate, like in fish, wich is made by feed or dripping in the water. The knowledge of homeopathy is wide, and involves: physics concepts, like the ultradilution concept, drug action in non-existing molecular of the drug itself; biology concepts, approaching the dynamic of the function in organisms, as like as the interaction of life with physic-chemical environment; and medicine concept, applying the physical, mental, psychological and social welfare / Mestre

Made available in DSpace on 2014-06-11T19:27:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-28Bitstream added on 2014-06-13T19:07:01Z : No. of bitstreams: 1 000748629.pdf: 1457578 bytes, checksum: c75b51fdc7faf4a8fe4a16a30730afe6 (MD5) / Neste trabalho foi registrada a primeira ocorrência de infecção por Citrobacter freundii, Lactococcus lactis subsp. lactis e Enterococcus faecalis associadas a surtos de doenças em bagres Sul Americanos. Além do isolamento das bactérias, também foi realizada a caracterização bioquímica e o perfil de susceptibilidade a antimicrobianos dos isolados. Para o isolamento, foram utilizados bagres provenientes de surtos em diferentes fazendas-berçário no Estado do Mato Grosso do Sul e também de criações em laboratório. Os peixes com sinais clínicos de bacteriose foram submetidos à necropsia e biópsia de rim e encéfalo para isolamento bacteriano em meios de cultura apropriados. Os isolados obtidos foram submetidos à coloração de Gram, teste da oxidase, catalase, produção de hemólise e identificação presuntiva através de kits comerciais API 20E ou API 20 Strep. A análise de susceptibilidade a 15 antimicrobianos foi realizada pelo método da difusão em disco com o Agar Muller Hinton. Foram obtidos dois isolados de C. freundii proveniente de cachara, que apresentaram multiresistência a 66,7 % dos antimicrobianos testados. Nos ensaios de infecção experimental, a doença foi reproduzida com sucesso, caracterizada como septicemia hemorrágica, com enterite acentuada e com reisolamento de C. freundii dos tecidos acometidos. Outros 57 isolados obtidos do rim e do encéfalo da cachara foram caracterizadas como cocos gram positivos associadas a meningoencefalite, identificadas como Lactococcus lactis subsp. lactis e Enterococcus faecalis. Todos os isolados apresentaram multiresistência aos antimicrobianos testados. A bactéria L. lactis subsp. lactis foi sensível somente a ampicilina e penicilina. Todas nossos isolados de E. faecalis foram sensíveis a sulfazotrim e a amoxilina e ao ácido clavulânico / The first occurrence of infection by Citrobacter freundii, Lactococcus lactis subsp. lactis and Enterococcus faecalis associated with outbreaks of diseases in South American catfish was recorded in this study. The isolates were biochemically characterized and the profile antimicrobial susceptibility performed. For isolation, fingerlings of catfish from sequential outbreaks at different farms nursery in the state of Mato Grosso do Sul and also creations in the laboratory were used. Fingerlings with clinical signs of bacterial infection were necropsied and biopsy of the kidney and brain obtained for bacterial isolation in appropriate culture media. The isolates were subjected to Gram staining, oxidase and catalase test, hemolysis production and presumptive identification using commercial kits API 20E and API 20 Strep. The analysis of susceptibility to 15 antimicrobials was performed by the disc diffusion method with the Muller Hinton Agar. Were obtained two isolates of C. freundii from cachara, which showed multiresistance to 66.7% of the antimicrobials tested. In experimental challenge, the disease was successfully reproduced, characterized as hemorrhagic septicemia, with severe enteritis, and reisolation of C. freundii from infected tissue was obtained. Another 57 isolates obtained from kidney and brain of catfish were characterized as gram-positive cocci associated with meningoencephalitis, identified as Lactococcus lactis subsp. lactis and Enterococcus faecalis. All isolates showed multidrug resistance to antimicrobials. The bacteria L. lactis subsp. lactis was only sensitive to ampicillin and penicillin. All our isolates of E. faecalis were sensitive to sulfazotrim and amoxilin and to clavulanic acid

Orientador: Maria José Tavares Ranzani de Paiva / Banca: Eduardo Makoto Onaka / Banca: Antenor Aguiar Santos / Banca: Paulo Teixeira Lacava / Banca: Ricardo Carneiro Borra / Resumo: Identificou-se no presente estudo o agente patogênico causador de surtos de estreptococose em cachara e surubins como sendo a Lactococcus garvieae. Considerando que a lactococose é uma doença emergente de importância econômica mundial, que gera surtos de mortalidade em massa em diversas espécies de peixes, avaliamos a possibilidade de prevenção e controle desta patogenia com o uso de vacinas. Após confirmação da patogenicidade da bactéria pelo Postulado de Koch, foram formuladas duas bacterinas inativadas com formalina, uma aquosa e uma oleosa, com emprego de adjuvante. Surubins híbridos foram imunizados via intraperitoneal com ambas bacterinas e com PBS. Para avaliar a eficiência das vacinas foi comparada a potência dos imunobiológicos desenvolvidos na sobrevivência dos animais, na cinética de anticorpos 15, 30 e 60 dias após a vacinação e na modulação da resposta imune após desafio bacteriano. Melhores níveis de proteção foram observados com a imunização com bacterina oleosa, onde foi observado maior nível de anticorpos circulantes e cujo valor de porcentagem relativa de sobrevivência foi de 81,7%. O presente fez o primeiro registro de surto de septicemia hemorrágica causada por Lactococcus garvieae em surubins híbridos e cacharas, e demonstrou que a imunidade dos surubins pode ser estimulada com emprego de vacinas, e que a bacterina oleosa proporciona proteção mais eficaz contra a lactococose / Abstract: The present study identified the pathogen causing outbreaks of estreptococose in cachara and surubins as being the Lactococcus garvieae. Whereas lactococose as an emerging disease of worldwide economic importance, that generates outbreaks of mass mortality in several species of fish, we evaluated the possibility of prevention and control of this pathogen with the use of vaccines. After confirming the pathogenicity of the bacterium by Koch postulate, two inactivated bacterins with formalin, an aqueous and an oily, with the use of adjuvant, were formulated. Surubins hybrids were immunized intraperitoneally with PBS and both bacterins. To evaluate the efficacy of the vaccines was compared the potency of the biopharmaceuticals developed in survival of the animals, on the kinetic of antibody 15, 30 and 60 days after vaccination and in the modulation of the immune response after bacterial challenge. Best levels of protection were observed with immunization with oily bacterin, where were observed higher level of circulating antibodies and whose value relative survival percentage was 81.7 %. This work made the first recorded outbreak of haemorrhagic septicemia caused by Lactococcus garvieae in hybrid surubins and cacharas, and demonstrated that the immunity of surubins can be stimulated through the use of vaccines, and oily bacterin provides more effective protection against lactococose / Doutor

by Wong Pik-kwan. / Thesis submitted in: September 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 116-139). / Abstract also in Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENT --- p.iii / TABLE OF CONTENTS --- p.iv / LIST OF TABLES --- p.viii / LIST OF FIGURES --- p.ix / Chapter CHAPTER ONE --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER TWO --- LITERATURE REVIEW / Chapter 2.1 --- Introduction --- p.4 / Chapter 2.2 --- Heavy metals --- p.6 / Chapter 2.3 --- Mechanisms of metal toxicity --- p.9 / Chapter 2.4 --- Toxic effects of metals on marine organisms --- p.10 / Chapter 2.4.1 --- Cadmium --- p.10 / Chapter 2.4.2 --- Chromium --- p.10 / Chapter 2.4.3 --- Copper --- p.11 / Chapter 2.4.4 --- Lead --- p.12 / Chapter 2.4.5 --- Nickel --- p.12 / Chapter 2.4.6 --- Zinc --- p.13 / Chapter 2.5 --- Metal uptake and elimination in marine organisms --- p.14 / Chapter 2.5.1 --- Uptake of metals --- p.14 / Chapter 2.5.2 --- Elimination of metals --- p.15 / Chapter 2.5.3 --- Metal detoxification system in fish --- p.16 / Chapter 2.6 --- Heavy metals in marine fish --- p.17 / Chapter 2.7 --- Bioaccumulation --- p.20 / Chapter 2.7.1 --- Models of metal accumulation --- p.21 / Chapter 2.7.2 --- Compartment model --- p.21 / Chapter 2.7.3 --- Physiologically based pharmacokinetic (PB-PK) model --- p.22 / Chapter 2.8 --- The influence of environmental factors on bioaccumulation of metals --- p.23 / Chapter 2.8.1 --- Temperature --- p.23 / Chapter 2.8.2 --- Salinity --- p.23 / Chapter 2.8.3 --- Organic matter --- p.24 / Chapter 2.8.4 --- pH --- p.25 / Chapter 2.8.5 --- Chelators and surfactants --- p.25 / Chapter 2.8.6 --- Other metals --- p.26 / Chapter 2.9 --- Biological effects of heavy metals on man --- p.26 / Chapter 2.10 --- The use of biological indicator organisms for metal pollution --- p.28 / Chapter CHAPTER THREE --- HEAVY METAL CONCENTRATIONS IN CULTURED MARINE FISH IN HONG KONG / Chapter 3.1 --- Introduction --- p.31 / Chapter 3.2 --- Materials and methods --- p.36 / Chapter 3.2.1 --- Sampling --- p.36 / Chapter 3.2.2 --- Water analysis --- p.36 / Chapter 3.2.3 --- Sediment analysis --- p.39 / Chapter 3.2.4 --- Mussel analysis --- p.40 / Chapter 3.2.5 --- Fish analysis --- p.40 / Chapter 3.2.6 --- Quality control and statistical analysis --- p.41 / Chapter 3.3 --- Results --- p.42 / Chapter 3.3.1 --- Seawater --- p.42 / Chapter 3.3.2 --- Sediment --- p.46 / Chapter 3.3.3 --- Mussel --- p.46 / Chapter 3.3.4 --- Fish --- p.50 / Chapter 3.4 --- Conclusion --- p.67 / Chapter 3.4.1 --- "Metal concentration in seawater, sediment, green mussel and fish" --- p.67 / Chapter 3.4.2 --- Accumulation of heavy metals in different tissues of cultured fish --- p.69 / Chapter 3.4.3 --- Relationship between the body weight and metal accumulation --- p.71 / Chapter 3.4.4 --- Heavy metal pollution in fish culture sites --- p.72 / Chapter 3.4.5 --- Selection of fish culture site --- p.72 / Chapter CHAPTER FOUR --- ACUTE AND SHORT-TERM EFFECTS OF COPPER(II) IONS ON SPARUS SARBA / Chapter 4.1 --- Introduction --- p.76 / Chapter 4.2 --- Materials and methods --- p.79 / Chapter 4.2.1 --- Experimental animals --- p.79 / Chapter 4.2.2 --- Determination of the 96 hour median lethal concentrations --- p.19 / Chapter 4.2.3 --- Determination of growth rate --- p.80 / Chapter 4.3 --- Results --- p.82 / Chapter 4.3.1 --- Determination of the 96 hour median lethal concentrations --- p.82 / Chapter 4.3.2 --- Determination of growth rate --- p.82 / Chapter 4.3.3 --- Distribution of Cu concentration in whole body and different tissues of S. sarba --- p.82 / Chapter 4.4 --- Conclusion --- p.91 / Chapter 4.4.1 --- Determination of the 96 hour median lethal concentrations --- p.91 / Chapter 4.4.2 --- Determination of growth rate --- p.93 / Chapter 4.4.3 --- Distribution of Cu concentration in whole body and different tissues of S. sarba --- p.94 / Chapter CHAPTER FIVE --- ACCUMULATION AND ELIMINATION OF COPPER(II) IONS TO SPARUS SARBA / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.2 --- Materials and methods --- p.98 / Chapter 5.2.1 --- Experimental animals --- p.98 / Chapter 5.2.2 --- Uptake and elimination of Cu ion in S. sarba during continuous exposure to waterborne Cu --- p.98 / Chapter 5.3 --- Results --- p.100 / Chapter 5.4 --- Conclusion --- p.108 / Chapter CHAPTER SIX --- GENERAL CONCLUSION --- p.112 / CHAPTER SEVEN REFERENCES --- p.116

The bacterial pathogens Streptococcus agalactiae and S. iniae have the capacity to infect a wide range of fish species throughout the world, with Nile tilapia (Oreochromis niloticus) being particularly susceptible. Global tilapia aquaculture production was estimated to be 3.5 million tonnes in 2008, and has a significant contribution in the global farmed fish market. Due to their ability to adapt to a wide range of culture systems the commercialisation of tilapia production has occurred in more than 100 countries. However, countries such as China have suffered from severe and extensive outbreaks of streptococcosis in cultured tilapia continuously for many years. Such large-scale outbreaks in China have resulted in a loss of approximately US$0.4 billion in 2011. Fish are permanently exposed to a plethora of pathogens and natural disease outbreaks are complex host-pathogen interactions that seldom involve single pathogen infections. As a consequence, simultaneous infections, alternatively called concurrent or co-infections, are starting to receive interest from aquatic disease researchers. Streptococcus agalactiae and S. iniae infections can both occur in the same geographic area and both S. agalactiae and S. iniae have been found to be present on the same farm in a single disease outbreak. It has been found that a disease outbreak caused by one these pathogens can be followed by another outbreak from the other. These two pathogens have serious effects on the tilapia aquaculture industry yet there is no information regarding S. agalactiae and S. iniae co-infections. Such information would be valuable for understanding epidemiology and the development of improved treatment and control of aquatic streptococcosis infections. The overall aim of this study was to investigate the pathogenesis of S. agalactiae and S. iniae in Nile tilapia. One important aspect of investigating simultaneous infections was to examine if there was any competition or synergy between S. agalactiae and S. iniae in vitro or in vivo. It was found that competition between S. agalactiae and S. iniae in vitro was inconsistent between different experimental systems. Results indicated that there was either no interaction between bacterial species or they coexisted during in vitro competition assays. Whereas, an in vivo model utilising wax moth larvae (Galleria mellonella) suggested that during a simultaneous infection with S. agalactiae and S. iniae the total levels of larval mortality were lower than expected indicating that the pathogens may have interacted with one another in a competitive manner. Investigations were also conducted to identify the expression of virulence factors in vitro for S. agalactiae and S. iniae. Comparisons were then made to ascertain any inter- and intra-species variation. Results demonstrated that both S. agalactiae and S. iniae strains possessed a capsule but varied in their haemolytic activity, blood survival and resistance to complement-mediated killing. These variations suggested that the two bacterial species differed in their mechanisms of pathogenicity where aquatic S. agalactiae strains may initially have a more systemic spread of infection and aquatic S. iniae strains may utilise a more localised spread of infection within the host. This hypothesis was tested through the development of a robust and reliable challenge model for S. agalactiae and S. iniae in Nile tilapia. Through this work it was apparent that fish infected with S. iniae experienced an acute infection with morbidity/mortality occurring 1 – 3 days after exposure. Whereas, the S. agalactiae challenged fish showed a more chronic infection with morbidity/mortality occurring from 1 – 6 days after exposure. Findings clearly demonstrated a more systemic spread of infection during a S. agalactiae challenge with high bacterial loads in all the organs examined. Streptococcus iniae was observed in fewer organs of infected fish and bacterial numbers were substantially lower. Concurrent infections are complex in natural conditions and in experimental studies. As a result a substantial amount of research will be required to fully understand the nature of co-infection with these two streptococci. This study has provided a solid foundation upon which to base future work.

The ectoparasitic monogenean fluke, Gyrodactylus salaris, is a parasite known to be highly pathogenic to Atlantic salmon (Salmo salar). Although present in the environment of several neighbouring European countries, the UK is thought to be G. salaris-free, but, if national contingency plans to control this parasite are to be effective, it is vital that we understand the factors underlying its transmission from host to host. This study demonstrates that the majority of parasites transferring to new hosts are mature parasites that have reproduced at least once. Since, exploration and host transfer strategies pose a risk to survival; the parasite will endeavour to pass on its genes before attempting to transfer from one host to another. This study has also shown that when pregnant parasites are forced to leave their hosts, their offspring are aborted prematurely to ensure the survival of the mature parasite. Gyrodactylids do not possess a free-swimming stage in their life cycle, which allows for their migration between hosts. In spite of this, they are able to rapidly colonise naïve hosts, even in non-shoaling populations of fish. This study investigates the transmission strategies employed by detached parasites in the colonisation of new hosts. Observations of gyrodactylids collected from 3-spine sticklebacks, Gasterosteus acuelatus, suggest that their activity increases as a stickleback approaches, alerting the host to its presence. The parasite is then ingested directly by the prospective host. A time series of experimental exposures and specimens prepared for Scanning Electron Microscopy (SEM) suggest that once ingested, the parasites attach to the lining of the buccal cavity and then migrate out to their preferred colonisation site on the outer surface of the fish. It is proposed that this may be an alternative route for host infection. Similarly, direct ingestion by the scavenging on infected hosts by 3-spine sticklebacks suggests another route of infection of new hosts. Although these routes of transmission may be of lesser significance, infections in the buccal cavity may be an important indicator for detection of infection and those personnel involved in screening fish for gyrodactylids should be aware that this is an area in which infections can occur. This study also demonstrated that the use of the anaesthetic 2-phenoxyethanol does not affect the number of gyrodactylids which leave the host to colonise a new host. Additionally, observations of the transmission process suggest that turbulence produced by the movement of the fish’s fins may facilitate the transfer of detached parasites from the substrate. While this hypothesis appears to be supported by video evidence and photographic stills gathered throughout the duration of this study, further work should be conducted using particle tracking techniques to determine the efficacy of using a vortex effect as a means of colonising new hosts. Field sampling processes may have an effect on this type of research, giving rise to problems with the accurate diagnosis, management and control of gyrodactylids in a variety of fish. Gyrodactylus infected specimens of 3-spine stickleback (Gasterosteus aculeatus L.), minnows (Phoxinus phoxinus L.) and stone loach (Barbatula barbatula L.) from one Scottish river were cohabited. The study found that small numbers of Gyrodactylus do transfer to atypical hosts. This study highlights that personnel involved in fish disease surveillance programmes should be aware of the consequences of transporting multiple species in the same transport vessel as gyrodactylids may infect species previously thought to be resistant. Equally, diagnosticians should be aware of the fact that atypical species may act as temporary hosts and that their gyrodactylid fauna should not be assumed. Non-feeding life-cycle stages, such as the dispersal stages of parasites, are dependant for survival upon finite energy reserves gathered during feeding phases. Thus, those individuals with more limited reserves will die sooner and consequently have less time available to find a new host once detached. At this stage, the principal energy reserves in gyrodactylids are stored as large lipids droplets. Confocal laser scanning microscopy (CLSM) has been used to investigate the distribution of lipid droplets in Gyrodactylus, which have migrated off their fish host, testing the hypothesis that these droplets function as a proxy for the nutritional state. This study, demonstrated that the lipid droplets were particularly associated with the gut and that there is a significant variability in the volume of stored lipid carried out by each individual. Transmission Electron Microscopy (TEM) showed that gyrodactylids carry lipid droplets at all stages of their life cycle, including at release from the birth pore. It is likely that transferring worms require stored energy reserves to survive in the event of failure to establish contact with a new host. These reserves could allow the parasite to survive without a host for several days. As gyrodactylids appear to respond to a range of stimuli including vibration and chemicals released from the host, the presence or absence of such cues may have consequences on the rates of Gyrodactylus transmission. If these chemical stimuli can be identified and then mimicked or blocked, then this may offer potential opportunities for the control of gyrodactylid behaviour and for disrupting their transmission to new hosts. Baseline gyrodactylid behaviour, in the absence of a host, was determined under white light and infrared. This was achieved using a specially constructed arena and purpose written image analysis software to analyse parasite movement under different lighting conditions. The study found that gyrodactylids were more active in the dark than in light conditions, typically displaying longer, more sinuous tracks under red light than under white light. To begin investigating the effect of chemical presence on gyrodactylid behaviour, the activity of octopaminergic agonists and antagonist which bind to muscle receptors and alter muscle activity, were assessed. The impact of octopamine hydrochloride, clonidine hydrochloride, amitraz and, a toxic reference, chlordimeform, over a range of concentrations (0.2 to 3.2µM/L) were assessed on gyrodactylid behaviour. All of the four chemicals affected Gyrodactylus and produced muscle tetanus, causing muscle spasms when extension was attempted. Prolonged exposure resulted in death. Only the highest concentration of chlordimeform, the toxic reference, affected 100% of Gyrodactylus after 24 hours. After 48 hours, all of the Gyrodactylus treated with chlordimeform were either affected, moribund or dead. Amitraz was more toxic than chlordimeform with 80% of Gyrodactylus being dead after 24 hours at the highest concentration. After 48 hours 100% of Gyrodactylus exposed to 3.2 µm/L amitraz were dead, and up to 80% were dead in those exposed to lower concentrations; with no parasites being left unaffected. Although these particular compounds are toxic to fish, the effect of these agonistic chemicals on Gyrodactylus behaviour and survival is interesting and suggests that a closely related compound that is safe for use against fish may offer a potential treatment for the control of G. salaris infections in rivers. An ultrastructure study was undertaken to contribute to the current understanding of gyrodactylid ultrastructure. The findings of this research require broad understanding of gyrodactylid behaviour for their interpretation. Photographic evidence was gathered using transmission and electron microscopy. From these results, it is clear that Gyrodactylus gasterostei on a three-spine stickleback host will respond to a range of stimuli (i.e. vibration or chemical cues released from the host) in their assessment of host suitability.

Tuberculosis (TB) is still a global epidemic disease despite its discovery over 100 years ago. It is caused by Mycobacterium tuberculosis, which invades and replicates within macrophages, key cells of the innate immune system. The hallmark of tuberculosis is the granuloma which is an accumulation of Mycobacterium-infected cells surrounded by immune cells, and the containment of the bacteria is assured as long as the host immune response remains intact. Despite a well-developed immune response in the infected host, reactivation of latent tuberculosis infection (LTBI) may occur through the introduction of other bacterial pathogens, re-infection with M. tuberculosis or due to other immunosuppression, e.g. AIDS or cancer. The zebrafish–M. marinum model provides an ideal system for examining the pathogenesis of tuberculosis and the associated immune response of the host due to its vertebrate-like immune system, and the close phylogenetic relationship of M. marinum to M. tuberculosis. Granuloma formation and immune response to M. marinum have been investigated mainly in zebrafish embryos or larvae, which lack an adaptive immune response, and little work has been performed in adult fish. This complicates the transfer of findings in these models to chronic, latent or re-activated disease stages in humans, where adaptive immunity plays an important part. The aim of the research presented here was to investigate the immune response of the adult zebrafish to M. marinum infection, with the focus on the kidney as one of the major immune organs in fish. The results obtained support further use of the adult zebrafish-M. marinum model for human tuberculosis infections in the future. In the present study, adult zebrafish were infected with low doses of M. marinum (NCIMB 1297 or NCIMB 1298) and the kidney was investigated for histopathological changes in the form of granulomas over a period of two months(Chapter 3). No granulomas were detected in the fish infected with M. marinum NCIMB 1298 while in zebrafish infected with NCIMB 1297, macrophage aggregation and granuloma formation were detected as early as day 11 post-infection. Occurrence and severity of granulomas and the presence of replicating bacteria increased over time, resulting in a high density of non-caseating and caseating granulomas in the head and posterior kidney after two months of infection. Interleukin 1 beta (IL-1β), Interleukin-12 (IL-12), Tumor necrosis factor alpha (TNFα) and Interferon gamma (IFNγ) have been shown to be important cytokines functioning in defence against tuberculosis, especially IFNγ which is considered to play an important part in acute, chronic and latent tuberculosis. Changes in gene expression of these immune genes in adult zebrafish were investigated over the first two weeks of infection with M. marinum NCIMB 1298 and NCIMB 1297. The results obtained in the first week after infection were inconclusive for both strains investigated. In agreement with the results presented in Chapter 3, no specific immune response was detectable in fish infected with M. marinum NCIMB 1298. However, after 14 days, a high-fold change in IL-12 and TNFα expression were detected in fish infected with M. marinum NCIMB 1297, while IL-1β showed no changes compared to the control fish. Furthermore, no IFNγ expression was detectable over the first two weeks of infection. The delay in the expression of IL-12 and the lack of IFNγ expression can be explained by the ability of M. marinum to manipulate the host immune response, as described for M. tuberculosis and other intracellular bacteria. Besides in vivo investigations of the host-pathogen interactions, in vitro primary macrophage cultures from individual zebrafish kidneys were developed to investigate macrophage-specific gene expression to M. marinum infection (Chapter 4). Although the results looked promising, further optimization is required before the results of the in vitro assays can be fully compared to the in vivo results. Our understanding of reactivation in latent tuberculosis infection (LTBI) both in healthy and immune compromised individuals is insufficient and is delaying the development of treatments for the disease. Therefore, the transcriptome profile of long-term infections (26 weeks) with M. marinum NCIMB 1297 in adult zebrafish was investigated to determine whether the gene expression in this model is comparable to LTBI in humans or other vertebrate model organisms (Chapter 5). In addition, transcriptome profiling was investigated in a group of long-term infected zebrafish exposed to stress to induce re-activation of the disease. Expression profiles in the long-term infected fish and the infected plus stressed fish differed from each other and displayed similar gene profiles to those found in the latent or re-activated disease states, respectively, in human and other vertebrate models. Infected fish displayed a profile highlighted by IFNγ, TNFα, NOS2b and IL-8 expression alongside activating and regulatory T cell responses, including involvement of cytotoxic T cells (CTLs). The transcriptome profile of the group of fish that had been infected and then stressed was distinguished by the lack of IFNγ expression and reduction in TNFα and NOS2b expression, as well as a lack of T cell response compared to the infected fish. In conclusion, the results obtained from Chapters 3 and 4 showed that M. marinum NCIMB 1298 is non-pathogenic to zebrafish. Infection with M. marinum NCIMB 1297, on the other hand, resulted in a similar immune response to that described for human and other mammalian vertebrate models (Chapters 3-5). These results support the use of the adult zebrafish-M. marinum model to investigate LTBI and disease reactivation, and will aid our understanding host-pathogen interactions for tuberculosis in the future.

The Scottish salmon industry is facing challenges in the control of aquatic infectious disease, as is the case in other countries such as Chile and Norway. Disease outbreaks can have an enormous economic impact and possibly affect wild fish populations. Disease transmission in an aquatic environment is complex and there are several transmission routes (vertical transmission, natural reservoirs, hydrodynamic transmission and long-distance movements). Effective control methods such as vaccines are not available in all cases and therefore disease prevention remains a priority. In livestock, epidemiological network models have been proven to be a highly useful tool to investigate the role of different transmission routes on the course of epidemics and have the potential to provide the same utility for aquatic networks. Understanding the complex contact network will result in more effective disease prevention, surveillance systems and control strategies. The aim of this thesis was to investigate the Scottish live fish movement network and its consequences for pathogen transmission between farms in order to develop and optimize control strategies for epidemics. The main objective of chapter 3 was to investigate the effect of different fallowing strategies on the spread of diseases with different transmission properties. A network model was constructed that included both local transmission and long-distance transmission. The basic structure of this network was a ring model where neighbours within a management area could infect each other and non-local transmission occurred at random. The results showed that when long-distance transmission was under reasonable control in comparison with local transmission risk, synchronized fallowing at the management area level was potentially a highly effective tool in the control of infectious diseases. Chapter 4 presents a detailed description of the number of live fish movements and their timing for Atlantic salmon (Salmo salar) in Scottish aquaculture. For this, movement records from 2002 to 2004 were provided by Marine Scotland, Aberdeen. Salmon are anadromous and have a freshwater (FW) and seawater phase (SW). Scottish live fish movements can be divided in FW-FW, FW-SW, SW-SW, SW-FW and “other” movements. The latter are mainly movements from and to research sites. This study showed that the contact structure and timing of live fish movements are seasonal and differ largely between production phases. Disease control measures should take these differences into account to optimize their strategies. In chapter 4, live fish movements were shown to be seasonal; therefore in chapter 5 the main aim was to quantify the effects of seasonality of live fish movements on the course of epidemics. The results showed that the sequence of salmon movements is important for the course of an epidemic. Seasonality is important when local transmission is higher than 0.05 per contact per week and when the movements are not clustered and when movements do not occur in a specific order based on the specific assumptions made in this model. In conclusion, this thesis described the complex live fish movement structure of salmon in Scotland and showed that biosecurity in SW farms is good but could be further improved if all management areas apply synchronized fallowing. The results of this study suggest that biosecurity between freshwater sites could be improved by the application of a system similar to management areas in SW farms.