Several systems were examined for the production and delivery of recombinant vaccines for fish. C. crescentus was employed to produce a fragment of the IHNV glycoprotein. When administered by injection to 0.5 gram rainbow trout (Oncorhynchus mykiss), one of the fusion proteins (184 amino acids of the IHNV glycoprotein fused to 242 amino acids of the C-terminus of the Caulobacter crescentus) protected the fish against lethal challenge with IHNV. Attenuated strains of Yersinia ruckeri were generated using allelic exchange mutagenesis. These strains were characterized in terms of in vitro growth characteristics and invasiveness. Attenuated E. coli and Y. ruckeri were exploited to deliver plasmid DNA to fish cells in vitro; attenuated Y. ruckeri bacteria were examined in vivo as bivalent vaccine delivery vehicles, either through the expression of a fragment of the IHNV glycoprotein or by carrying a plasmid DNA vaccine encoding the complete IHNV glycoprotein. A cell wall deficient strain (11.29��dap) protected rainbow trout against lethal challenge with pathogenic Y. ruckeri. Gene transfer to fish was not detected by luciferase reporter gene assays. No clear protection from IHNV challenge was observed. / Graduation date: 2002

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 130-160).

A series of experiments with Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV) were carried out to determine dynamics of the spread of infection in chinook salmon (1.2-1.98g) and rainbow trout (1.2-3.1g). It was found in experiments with A. salmonicida that fish infected by bath immersion became infectious at 4 days postexposure (dpe), one day prior to dying from furunculosis. In cohabitation experiments with a single infected fish donor, an average of 75% disease specific mortality was obtained. There was suggestive evidence that there is a positive relationship between holding volumes and furunculosis prevalence in cohabitation experiments with single donor fish. Median day to infection was inversely correlated with density. The threshold density at density of 1.97 fish/L was approximately 30 times less than the density of 0.08 fish/L, 13.33 and 320 fish respectively. Reproductive ratio (R₀) and transmission coefficient (β) in the furunculosis epizootic were 3.23 and 0.021 (individuals*day)⁻¹ respectively. The mortality rate (α) of infected animals was 28.7% per day. The models constructed successfully mirrored the results of laboratory experiments. Data produced by simulation of the models were significantly associated with the data obtained from laboratory experiments for susceptible (S) class and also for infected class. In similar experiments carried out with IHNV, it was found that donor fish became infectious 3 dpe. The virus levels in donor fish and prevalence levels were also highly associated. Smaller volumes of that led to higher prevalence levels than observed in bigger volumes with 60 or 30 fish in each. A single donor fish was able to transfer virus to recipient fish. However, unlike the A. salmonicida experiment, transmission was insufficient to initiate a full-scale infectious hematopoietic (IHN) epizootic. Estimated parameters for dynamics of infection were approximately half of the values for A. salmonicida (R₀=2.57,β=0.008 (individuals*day)⁻¹ and α=0.15). The models constructed for IHNV spread were used to simulate the results of density experiment. However, it was not possible to test the association between susceptible and infected classes due to inadequate number of infected fish. / Graduation date: 2001

Submitted by (edna.saturno@ufrpe.br) on 2017-02-22T12:37:59Z No. of bitstreams: 1 Suely Santos Bezerra.pdf: 418523 bytes, checksum: 6cbf43e2cf1c398824dd661f7ea8c038 (MD5) / Made available in DSpace on 2017-02-22T12:37:59Z (GMT). No. of bitstreams: 1 Suely Santos Bezerra.pdf: 418523 bytes, checksum: 6cbf43e2cf1c398824dd661f7ea8c038 (MD5) Previous issue date: 2007-02-23 / It can be observed nowadays that the increase in the commercial shrimp produc-tion occurs concomitantly with an enhancement of the incidence of diseases. These last ones can cause innumerable damages to the shrimp farmers in the whole world. Amongst these diseases, it can be distinguished the Infectious Hypodermal and Hema-topoietic Necrosis (IHHN), with viral etiology, that can be characterized by not causing great mortality, but body deformities. In addition to this, it frequently leads shrimp to heterogenic growth. For viruses diagnosis histology is an efficient tool, allowing the verification of the health of these crustaceans. This study aimed the observation of the occurrence of IHHN characteristic histopatologic injuries in Litopenaeus vannamei, and its relation with parameters of culture of marine shrimp farms in the state of Per-nambuco. After multiple regression statistic analysis it could be concluded that the tem-perature of the water of the pound and its oxygen availability are narrowly related with the occurrence of those histopatologic injuries. / Ao mesmo tempo em que crescem os cultivos comerciais de camarão, observa-se o aumento da incidência de enfermidades que podem causar inúmeros prejuízos aos carcinicultores em todo o mundo. Dentre essas enfermidades, destaca-se a Infecção Hi-podermal e Necrose Hematopoiética (IHHN), de etiologia viral que, embora não cause grandes taxas de mortalidade, produz retardo no desenvolvimento e deformidades nos camarões. Para o diagnóstico dessa virose a histologia tem sido uma ferramenta bastante eficiente e que permite maior monitoramento da sanidade desses crustáceos. Desta for-ma, objetivou-se estudar a IHHN em Litopenaeus vannamei relacionando lesões histo-patológicas características e sua relação com variáveis de cultivo. Com base em regres-sões múltiplas conclui-se que existe uma alta relação entre as variáveis de cultivo tem-peratura e oxigênio e a ocorrência destas lesões.

This dissertation describes two different projects. The first is the development of an oral DNA vaccine delivery system for fish. A novel oral DNA vaccine delivery system was developed for Rainbow Trout by combining non-viral vectors (polycationic liposomes or polycationic polymer) to facilitate the DNA vaccine's uptake by cell membranes along with enteric-coated protection of the DNA embedded in microparticles to prevent DNA degradation in the gastrointestinal tract. Spray drying and spray coating bead techniques were employed in the preparation of the DNA vaccine microparticles. The spray drying technique allowed production of spherical shape enteric-coated microparticles with a particle size range of 0.18 to 20 ��m. Larger particle sizes of 40-50 mesh were obtained from the spray-coated bead technique. The resultant DNA vaccine microparticles were granulated with regular fish feed and given to fish to investigate the efficacy of the delivery system in providing protection against IHNV, and to demonstrate the ease of administration in fish. An in vivo fish trial experiment showed improvement in fish survival rate when fish were immunized with larger particle size DNA vaccine microparticles. Further research to find effective vector carriers for the DNA vaccine delivery system and to seek modifications of the delivery system that will still prevent the denaturation of plasmid DNA that will also facilitate membrane uptake of the DNA vaccine is needed in order to develop a safe, effective, and commercially viable vaccine to control the outbreak of IHNV. The second project of the dissertation is prediction of in vitro drug release profiles from a novel matrix tablet spray-coated with a barrier membrane using mathematical and statistical models. Tablets were prepared by direct compression followed by spray coating. The relationship of the amount of hydrophilic materials in the core tablets and barrier thickness on drug release mechanism was investigated using factorial design and regression analysis. Drug release characteristics were influenced and can be controlled by modifying the amount of hydrophilic materials in the core tablet and the barrier thickness. Mathematical equation generated from regression analysis of n-value, lag time, and percent drug release as a function of the amount of hydrophilic material and the amount of coating material applied can now be used as a tool for predicting and optimizing in vitro drug release from matrix tablets spray-coated with a barrier membrane. / Graduation date: 2003