Dissertations / Theses on the topic 'Fish diseases'

The prevalence and impact of proliferative kidney disease (PKD) and myxosporidiosis has been investigated in wild fish stocks in the UK, over 1,500 fish representing 17 species being examined. PKD was recorded in brown trout, grayling and pike, the causative agent, the PKX cell, being identified with the aid of light and electron microscopy. A further 27 myxosporean species were also noted, with Myxobolus cotti (syn. M. jiroveci), in the brain of bullheads Cottus gobio being recorded for the first time. Studies on the structure and development of Myxidium lieberkuehni in pike revealed several previously undescribed features. Comparative morphological studies were undertaken to assess affinities of PKX with known myxosporean species. Results indicated similarities with early presporogonic stages of several myxosporean species, especially those belonging to the genus Sphaerospora. The apparent rarity of spore formation associated with PKX infections in the hosts examined focussed attention on species of Sphaerospora as possible sources of infection to salmonids. Studies concentrated on the possible involvement of the 3-spined stickleback, Gasterosteus aculeatus and its renal parasite, Sphaerospora elegans, in PKD transmission. A re-description of this parasite (recently elevated to "type species" for the genus), was prepared. Laboratory experiments using rainbow trout PKX cells successfully transmitted the infection to rainbow trout, brown trout, brook trout and grayling; however sticklebacks challenged with PKX cells did not appear to become infected. Rainbow trout challenged with S. elegans spores and presporogonic stages showed no evidence of sphaerosporosis or PKD. Experiments designed to investigate the possible role of tubificid worms in PKD transmission provided inconclusive results. Field studies provided data on the pathogenesis of PKD in grayling and showed this species to be highly susceptible to the disease.

Mycobacterium spp. isolated from food and ornamental fish in Thailand (strains TB 1, TB40, TB267, TB268), and the type strains Mycobacterium marinum (NCIMB 1298), Mycobacterium fortuitum (NCIMB 1294), and Mycobacterium chelonae (NClMB 1474) were cultured in Long's medium, Eagle's minimum essential medium, Sauton's medium and modified Sauton's medium. The latter enabled excellent growth and production of extracellular products (ECP) from TB40, TB267, TB268 and M marinum in particular, whereas growth and production of ECP for all strains was limited in Long's medium. SDS-PAGE protein profiles of ECPs from 14 day culture supernatants showed major bands at 65, and <14 kDa. After 2 days culture at the higher temperature of 37°C (heat shock), the production of ECP from all mycobacteria strains except M marinum averaged approximately 4 to 10 fold higher than from strains cultured for 14 days at 28°C. The major fibronectin binding proteins from ECP of Mycobacterium spp. isolated from infected fish were identified at 21-25 kDa. Cross reactivity was detected between ECP from Mycobacterium spp. and MAb anti-heat shock protein (60 kDa) and MAb anti-M Tuberculosis. The 65 kDa antigen of TB267 is a strongly immunogenic protein eliciting antibodies in fish, rabbits and mice. Cross-reactivity was found between rabbit anti-65 kDa antibody and sonicated proteins from many other bacterial species. Therefore, the 65 kDa protein from Mycobacterium sp. isolated from snakehead fish may be a common protein in fish bacterial pathogens. Eighteen MAbs to TB 267 and M chelonae were produced. The epitopes to which the MAbs are against located on molecules susceptible to protease treatment. All MAbs recognized the 65 kDa protein. It is one of major proteins in the ECP, whole cell sonicates and lysates from Jv1ycobacterium spp. and is located in the peri plasmic space or cell wall, and is secreted in the medium during culture. A pnmary intraperitoneal (IP) immunisation of extracellular products (ECP) from Jv1ycobacterium spp., (strains TB40, TB267 or M marinum) mixed with Freund's incomplete adjuvant (FIA), followed by a secondary IP injection at 8 wks, resulted in the elevation of both the non-specific immune response (by measuring nitroblue tetrazolium, lysozyme and phagocytosis activity) and the specific immune responses of rainbow trout, Oncorhynchus mykiss (by measuring specific antibody levels). Nile tilapia were immunised by injecting extracellular products (ECP) of Jv1ycobacterium spp. (strain TB40, TB267 or the type strain M marinum) into their swimbladders and this resulted in the elevation of the non specific immune response. The cytological response of rainbow trout head kidney macrophages to ingested Mycobacterium spp was examined in vitro. The bacteria had previously been opsonised with either fresh rainbow trout serum (FS), or serum which had been heat-inactivated (IDS), or rainbow trout antiserum against the extracellular products (ECP) of Mycobacterium strains TB267 or M marinum. MAbs against the ECP were also used as opsonins. Opsonisation of the mycobacteria was found to greatly enhance the phagocytic and killing activity of the rainbow trout macrophage.

The pathogen Piscirickettsia salmonis, has been isolated from all species of salmonid and has been found in Chile, Canada, Ireland, Norway and Scotland. Rickettsia-like organisms from European sea bass (Dicentrarchus labrax) were found to share common antigens with the P. salmonis type-strain, LF-89 using the indirect fluorescent antibody test (IFAT) and immunohistochemistry (IHC). In addition, the DNA sequences of the 16S rDNA and 16S-23S internal transcribed spacer region (ITS) were compared with those published for P. salmonis strains and showed that the sea bass piscirickettsia-like organism (SBPLO) was another strain of P. salmonis, closely related to the salmonid pathogens. The ability of P. salmonis to survive and replicate within head kidney (HK) macrophages of rainbow trout infected in vitro was demonstrated using transmission electron microscopy (TEM) at various times post-infection (p.i.). However, macrophages derived from fish vaccinated against P. salmonis appeared to clear in vitro infection more rapidly than macrophages from naive fish. Polymerisation of filamentous actin within the cytoplasm of the host cell is used by some mammalian rickettsiae to achieve intercellular spread by actin-based motility (ABM). Both TEM and confocal microscopy were used to investigate possible actin tail formation by P. salmonis. No evidence of tail formation was found. Respiratory burst (RB) by P. salmonis was measured following exposure of rainbow trout HK macrophages to the organisms in vitro. Because of background stimulation of the RB by growth media and debris from the CHSE-214 cells used to culture P. salmonis, it was not possible to detect any effect of the pathogen on the burst. Schering Plough Aquaculture has developed a recombinant vaccine against P. salmonis. The ability of the vaccine to elicit a memory response against P. salmonis was investigated by measuring three different immune responses: a) the expression of iNOS was measured by reverse transcription polymerase chain reaction (RT-PCR) to detect mRNA levels or by the Greiss reaction to quantify the end-products of nitric oxide metabolism in the serum. Increased iNOS expression was not detected in rainbow trout kidney or serum following vaccination/challenge with P. salmonis. However, iNOS expression was detected in gill tissue from naive trout which suggests that expression may be constitutive in this tissue. b) the production of macrophage activating factor (MAF) by lymphocytes from vaccinated trout, following stimulation in vitro with P. salmonis, was measured by the ability of supernatants from these cells to prime elevated RB in naive macrophages. No difference in priming ability between supernatants from vaccinated and non-vaccinated fish was detected. However, macrophages among the immune leukocytes used to produce the MAF supernatants did exhibit elevated RB compared with macrophages from non-immune fish, suggesting that vaccination had produced a population of lymphocytes capable of priming activation of macrophages. c) by screening individual sera concurrently against the rickettsial and CHSE antigen preparations, the antibody response to P. salmonis could be detected specifically and was found to increase significantly in immunised fish by 6 weeks post-vaccination. Specificity of the response was demonstrated by screening the sera against Aeromonas salmonicida.

Peripheral blood leucocytes of the turbot, Scophthalmus maximus, were characterised into 4 distinct groups following morphological, morphometric and histochemical examination. Total and differential cell counts were determined. Thrombocytes, the most abundant leucocyte type (52%), were highly mobile and encountered in several morphological forms. Granulocytes, representing 5.6% of the leucocyte population, histochemically most resembled the mammalian neutrophil. Both large and small lymphocytes (40.8%), were encountered. Monocytes were rarely observed (1.6%). Thrombocytes and monocytes were phagocytic in vitro at 12oc and 22oc, showing increased phagocytic activity at the higher temperature. The thymus was paired and consisted of a well developed outer cortex and an inner meduallary region. The spleen was bounded by a fibrous tissue capsule and contained a large volume of blood. Diffuse areas of red and white pulp, ellipsoids and melanomacrophage centres were apparent. Lymphocytes, thrombocytes and mature erythrocytes made up the cellular components. The kidney, located beneath the vertebral column contained haemopoietic tissue throughout. Excretory tubules were evident posteriorly. Cellular elements included developing granulocytes, large and small lymphocytes and melanomacrophages. Investigation of ontogenic development of the lymphoid tissue, from 24h post-hatch to the completion of metamorphosis (Day 63) revealed thymic, splenic and kidney rudiments all present at Day 4 with the first lymphoid cells appearing in thymus and kidney by Day 8. Splenic lymphoid cells and the development of areas of white pulp were apparent by Day 28. Differentiation of the thymus had occurred and melanomacrophage centres were seen in the spleen, completing structural lymphoid development by Day 63. Critical stages of lymphoid ontogeny were correlated with easily recognisable external morphological features. A study of the kinetics of carbon clearance by the reticuloendothelial system, revealed a phagocytic capacity in the spleen, kidney and heart. Splenic carbon was seen at 20min post injection, accumulating around ellipsoids and rising to a maximum level at 24h. By Day 5 carbon levels within phagocytes, by now more distant from the ellipsoids, had begun to decrease and carbon was seen within melanomacrophages. Levels of kidney carbon, present within large macrophage-like cells which increased in size forming larger aggregations, increased to a maximum at Day 3. Clearance appeared more rapid in the posterior kidney. Low level uptake was seen within the epicardium. Carbon uptake was not observed in the liver or gill. Kidney leucocyte migration in vitro was examined to a range of chemoattractants using a number of assays. 24h bacterial culture supernatants of Vibrio alginolyticus induced significant cellular responses. The under agarose assay demonstrated migration inhibition to 100%, 50% and 40% supernatant dilutions. Enhanced migration was detected to dilutions of 5-50% in the microchemotaxis chamber, being optimal at 20%. The leucocyte polarisation assay demonstrated cell orientation in response to I 00% culture filtrate and the capillary tube migration assay revealed cellular inhibition at concentrations of 10% & SO%. Leukotriene B4 (LTB4) also induced migration in the filter-based assay, being optimal at to-7M. Cellular migration and orientation were observed in filter and polarisation assays to turbot serum, with normal and activated serum inducing elevated responses in the filter based assay. No response was detected by any of the assay systems to n-formylmethionyl-leucyl- phenylalanine (FMLP) or casein at any concentration tested. Results are discussed in relation to the cellular defence mechanisms of fish.

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.

In aquaculture worldwide, diseases are a significant constraint to economic expansion. The Scottish salmonid industry has experienced many cycles of development, with episodes of little or negative profitability caused by excess of production, and times of crisis due to different disease problems. In Scotland, the early implementation of regulation largely contributed to the control of infectious disease outbreaks. The recent Chilean outbreak of infectious salmon anaemia (ISA) illustrated the threats and the impacts of disease in the aquaculture industry and the importance of implementing good regulation and husbandry practices to reduce the impact of the spread of infectious disease. Databases of site production data have an important role to play in the investigation and understanding of diseases. They store valuable data collected during the time of production, which are essential for the identification of potential health and production problems during the production cycle of farmed fish. Mortality records are one of the most important sources of information on a farm, especially if it includes the cause of death as deformities, predators and diseases. Any deviation from the expected levels of mortality may indicate production problems, infectious diseases, or inadequate welfare. The investigation of increased rates of mortality must include examining farm records, determining the influence of death rate on production and the potential risk factors of diseases in a farm. This project demonstrated the importance of mortality records for setting industry standards of “expected” mortality losses and for investigating the value of recorded mortalities as a tool for aiding in surveillance and control of infectious diseases. It also aimed to determine the utility of reported mortality in supporting and assisting management-strategy decisions at the farm and industry level. In this project, we developed a baseline benchmark curve for expected mortality losses for Atlantic salmon in seawater. This novel approach constitutes a first attempt to establish a baseline curve for normal mortality, which allows detection of potential production problems based on deviations of mortality from the baseline curve of normal mortality. The results of this study also indicated that mortality levels may vary across production cycles, which can again be identified by using the baseline. We found that site was the factor with the highest contribution to variance in mortality. This site-to-site variation in mortality may have resulted from epidemics and environmental incidents, or other local event/effects. Temperature, and/or geographical area were also characteristics that contribute to variation in mortality. The regulator, Marine Scotland Science, with the backing and support of the salmonid industry has suggested potential mortality thresholds as an indicator of presence of infectious diseases, which could be used as alerts for inspection by the official authority. In this study, high mortality rates on fish farms were investigated as an indicator of the presence of infectious disease. The analysis was performed using several analytical approaches: receiver operating characteristic (ROC) curve analysis, measures of sensitivity and specificity, and bootstrap methods. The study was performed by splitting the production cycle into small fish with mean weight below 750 g and large fish with mean weight over 750 g. In the small fish, the results did not suggest reported mortality as a strong indicator of the presence of infectious disease, which may be caused by the lack of records of infectious disease at this stage of the production cycle. In the larger fish, high mortality rates were found to be a strong potential indicator of the presence of infectious diseases, including the suggested mortality threshold. In a survey, the role of traditional diagnosis in the prevention and control of disease outbreaks was assessed. For that, key informant interviews were performed with open questions to the health or farm manager of several trout and Atlantic salmon farms and we also used the diagnostic reports of the Veterinary Diagnostic Services (VDS) from Stirling University to triangulate the data. We showed that disease diagnoses are of great importance for disease identification and control of actual diseases. Farmer’s experience was also indicated as essential in the identification of the first signs of disease, which was principally through the daily monitoring of fish. This study suggested that disease diagnosis starts at the farm level with the daily monitoring of fish and the records of different parameters by the farmer, including mortality. Those records were showed to be vital to identify problems within the production. This thesis illustrated a novel approach to investigate and interpret recorded mortality at the farm level. The results presented in this thesis indicated reported mortality as a vital on-farm tool for identification of diseases and production problems. This thesis suggested priority areas where further investigation is required.

Vibrio anguillarum infects many fish species in aquaculture, reducing farm productivity and negatively impacting fish welfare. Deeper understanding of the biology of V. anguillarum, particularly during infections in vivo, will help to improve disease prevention and control. Thus, the aim of this thesis was to provide further insight into the infection biology of V. anguillarum with a view to identifying better ways to reduce the impact of this pathogen in aquaculture. Conventional studies on virulence, particularly those aiming to identify novel virulence factors, often employ transposon mutagenesis where the functions of individual genes in the bacterium are disrupted. These mutant libraries are screened to identify those with attenuated virulence, allowing subsequent identification of the gene responsible. Usually the native fish host would be used but such studies are increasingly difficult to perform due to regulations on vertebrate experiments and ethical concerns. As a result, alternative invertebrate hosts are now an important means to studying microbial infections, but few models have been assessed for bacterial pathogens of fish. In this thesis, larvae of the greater wax moth Galleria mellonella were evaluated as an alternative host to investigate V. anguillarum virulence. Wild-type V. anguillarum isolates killed larvae in a dose-dependent manner, replicated in the haemolymph, and larvae infected with a lethal dose of bacteria could be rescued by antibiotic therapy, thus indicating that V. anguillarum established an infection in G. mellonella. Crucially, virulence of 11 wild-type V. anguillarum isolates correlated significantly between larva and Atlantic salmon infection models, and studies with isogenic mutants knocked out for various virulence determinants revealed conserved roles for some in larva and fish infections, including the pJM1 virulence plasmid and rtxA toxin. Thereafter, 350 strains from a V. anguillarum random transposon insertion library were screened for attenuated virulence in G. mellonella. In total, 12 strains had reduced virulence and in these mutants the transposon had inserted into genes encoding several recognised and putative virulence factors, including a haemolytic toxin (vah1) and proteins involved in iron sequestration (angB/G and angN). Importantly, the transposon in one strain had inserted into an uncharacterised hypothetical protein. Preliminary investigations found this putative novel virulence factor to contain a GlyGly-CTERM sorting domain motif, with sequence similarity to VesB of Vibrio cholerae which is involved in post-translational processing of cholera toxin. Finally, three transposon insertion libraries were mass sequenced on a MiSeq platform to identify V. anguillarum genes lacking transposon insertions. These genes were assumed to be ‘required’ for viability in the conditions under which the mutants were selected, in this case tryptone soya agar. In total, 248 genes lacked a transposon insertion and were the putative ‘required’ genes, and these may be important chemotherapeutic targets for new approaches to combat V. anguillarum infections. This thesis has furthered our understanding of the biology of the important fish pathogen V. anguillarum using an ethically acceptable approach, and the findings may assist with new ways to reduce the burden of this bacterium in aquaculture.

A rainbow trout (Slamo gairdneri Richardson) population of IPN virus carriers was studied over a one-year period using both homogenisatian and co-cultivation for virus isolation. The percentage of virus-yielding fish was high between March and June but then declined. This was diametrically opposite to the trend in the serum antibody levels indicating that the marked humoral immune response resulted in a very significant reduction in the virus titres. The highest isolation rate was obtained from the kidneys after co-cultivation (from seventeen of the twenty-three virus-positive fish) underlining the very high sensitivity of this recently developed method for virus detection. Twelve of the twenty-three virus-positive fish yielded virus from the pyloric caeca after homogenisation. Virus was occasionally isolated from the faeces indicating that this may well be a possible avenue for horizontal transmission of the virus. No virus was ever detected in gonadal tissue. The virulence of the rainbow trout virus was enhanced in various ways and used to infect tilapia Oreochromis spilurus Gunther of different ages through a variety of routes. Fry infected by direct immersion, orally and by force feeding showed little or no signs of infection. Intraperitoneally and intramuscularly injected fingerling and adult fish developed marked haemorrhaging, severe loss of skin mucus and up to 50% mortalities were recorded. Gross pathology included enlarged and liquifying liver, gastroenteritis and mild brain haemorrhaging. Histopathologically there was extensive cellular vacuolation and degeneration as well as marked leucocytic infiltration in the liver, intestine and swimbladder. Eosinophilic granule cell infiltration of the intestinal wall was also very prominent. Virus was recovered from several organs and determination of virus titres revealed that active viral replication had occurred in the tilapia tissues, a finding further supported by electron microscopical evidence. The fish showed a clear humoral antibody response.

This project investigated several factors that influence the virulence of the fish pathogen Aeromonas salmonicida subspecies salmonicida. In this study the effect of iron supplementation was investigated on whole cell bacterin used as a vaccine. It was shown that A-layer negative strains grown under iron supplementation do not confer high protection. A-layer positive strains though, do provide protection at levels similar to the A-layer negative iron restricted bacterin. Use of a combined vaccine appeared to compound the protection. Cells grown under iron normal and iron restricted conditions have previously been shown to produce, respectively, ferric superoxide dismutase (SOD) and manganese SOD to protect against superoxide anions. Analysis here followed the transfer from iron restricted culture to iron supplemented culture to investigate the change in superoxide dismutase (SOD) induction. It appeared that both ferric and manganese SOD were expressed even though only one form was active in either iron condition. This implies post-translational control of activity. Catalase in another enzyme with a protective role and is induced by hydrogen peroxide. Cells grown without the addition of hydrogen peroxide do not appear to express a functional catalase. Analysis of catalase induction has shown that iron normal and iron supplemented growth using hydrogen peroxide induces expression of a catalase enzyme. Iron restricted bacteria show sensitivity to normal induction doses of hydrogen peroxide. Lowering the induction concentration allows expression of the catalase. This suggests that the catalase is heme co-factored. The relationship between iron levels and catalase induction suggests that catalase has a high priority for any available iron. The in vivo expression of the serine proteinase and iron regulated outer membrane proteins (IROMP's) was investigated. Whilst the serine proteinase was induced in vivo in a normal virulent strain, a proteinase-negative strain did not appear to produce the proteinase. This suggests that the serine proteinase is not essential for virulence. The expression of IROMP's in vivo indicates an iron restricted environment. A. salmonicada secretes a glycerophospholipid: cholesterol acyl transferase (GCAT) that is often complexed with LPS and believed to be a major toxin of the ECP. Analysis of culture supernatants has identified a secreted extracellular polysaccharide that may also complex with GCAT.

A standardised procedure was established for the laboratory maintenance of C.irritans in thick-lipped mullet. Nine isolates of C.irritans were obtained of which eight were successfully established for up to 48 weeks. Studies on individual life cycle stages with regards to longevity, viability, and susceptibility to a chemotherapeutic agent, revealed the difficulties in eradicating the cysts. Transmission of the parasite both to and from the host correlated with darkness. High infection levels resulted in the death of host fish within five days following exposure to theronts. An acquired protective immune response developed in host mullet within 14 days after exposure to sub-lethal infection. The degree of immunity appeared to relate to infection dose, and was not fully protective in all fish. Protection persisted for six months after infection and appeared specific to C.irritans. Specific antibodies to trophont antigen were identified in mullet serum but not epithelial mucus following either natural exposure to theronts or intraperitoneal immunisation with trophont antigens. Serum from intraperitoneally immunised fish caused theront immobilisation and agglutination in vitro; however no evidence was found for a protective role for specific antibody. Major polypeptides were identified and characterised by molecular weight for both trophont and theront stages using SDS-PAGE. Significant homology in major polypeptide profiles was found between C.irritans and I.multifiliis, in respect to trophonts and particularly theronts. Murine monoclonal antibodies raised to trophonts identified two polypeptide components of molecular weights 20-21kDa and 68-69kDa, the latter being homologous with host immunoglobulin heavy chain. These results are discussed in relation to future management and control strategies for cryptocaryosis in warmwater mariculture systems and aquaria.

The population ecology of the diseases of the common dab (Limanda limanda L.) has been studied and the use of fish disease as a monitor of pollution in the marine environment reassessed. The diseases included in this study were lymphocystis disease, hyperplasia/papilloma, ulcer disease, inflammatory fat-cell necrosis, and X-cell disease. The interactions responsible for determining disease abundance were assessed by carrying out a detailed longitudinal field survey and interpreting the observed changes in abundance by reference to the population biology of the common dab, the abiotic environment, and the known biology of the diseases. The diseases were not evenly distributed within the host population. Significant differences in prevalence occurred between sexes, ages, and fish of differing maturity status. These were interpreted in terms of varying susceptibility of the host, the response of the host to infection and the dynamics of the diseases. Analysis of the age-prevalence relationships suggests that although some diseases have the potential for their abundance to be regulated this is not fulfilled. The temporal distribution of the diseases consisted of long-term trends, seasonal cycles, and short-term fluctuations. A variety of potential causal factors were highlighted including the spawning behaviour of the dab, temperature, and bacterial abundance related to environmental productivity. The patterns in the spatial distribution of the diseases were reflected in variation in prevalence on both large (˜100nml) and small scales (˜1nml). In this case the causal factors highlighted were salinity, host population density, and again bacterial abundance related to productivity. No effect of pollution on disease prevalence was demonstrated. Neither could the occurrence of lymphocystis disease be related to the concentration of chemical contaminants in the livers of dab.

Three epitope-mapping procedures were used to identify B-cell epitopes on Betanodaviruses: neutralisation escape mutant sequence analysis, phage display, and pepscan. Betanodaviruses have emerged as major pathogens of marine fish. These viruses are the aetiological agents of a disease referred to as viral nervous necrosis (VNN), which affects many species of fish that are economically valuable to the aquaculture industry. The identification of betanodavirus B-cell epitopes will facilitate the rational development of vaccines to counter VNN. A panel of mouse monoclonal antibodies (MAbs) was produced using hybridoma methodology for use in each of the epitope mapping procedures. These antibodies were characterised in Western blotting, ELISA, and virus neutralisation tests. Rabbit polyclonal sera, and serum samples from nodavirus-infected fish were also used for pepscan analyses. Attempts to produce betanodavirus neutralisation escape mutants, using plaque assay or limiting dilution based methods, were not successful. Two phage libraries expressing random peptides of seven (Ph.D.7™) or twelve (Ph.D.12™) amino acids in length as fusions to the coat protein were used to identify the ligands recognised by MAbs directed against betanodavirus. Neither of these phage libraries yielded conclusive results. Phage clones containing tandem inserts were obtained after MAb selection from library Ph.D.7™. Extensive screening and nucleotide sequence analysis of MAb-selected clones from library Ph.D.12™) failed to yield a consensus sequence. Pepscan analyses were performed using the recently developed suspension array technology (SAT). This was used to map the recognition sites of MAbs and serum samples onto a panel of overlapping synthetic peptides (12mers) that mimicked the betanodavirus coat protein. The results of pepscan analyses required careful interpretation due to the binding of antibodies and serum samples to multiple peptides. However, three regions of the nodavirus coat protein were identified as containing B-cell epitopes: amino acids 1-50, 141-162, and 181-212. These results are discussed in relation to previous studies of immune responses to betanodaviruses, and to the future development of betanodavirus vaccines and diagnostic reagents.

Prior to 1989 the total number of Gyrodactylus species recorded for all British freshwater fish numbered 20. The fauna present on the British Salmonidae was poorly documented and frequently not identified to species level. The European free market, created in 1992, resulted in legislative changes allowing the movement of live fish stocks, albeit under strict disease monitoring conditions, into the UK. One stipulation maintains that the fish stock be free of the ectoparasitic monogenean Gyrodactylus salaris Malmberg, 1957, a parasite made notifiable in the UK in 1987 (Diseases of Fish Act, 1937) owing to its pathogenicity and damage to Norwegian salmon populations in 38 rivers. Although this parasite has been reported since 1957 throughout mainland Europe, its occurrence in the UK was unknown. This project set out to make a national survey of British salmon ids and investigated 250 sample sites, examined four salmonid hosts, Atlantic salmon Salmo safar, brown trout Salmo trutta, rainbow trout Oncorhynchus mykiss and Arctic charr Salvelinus alpinus. Seventy of the sites were found to be positive for Gyrodactylus. Distinctions were made between wild and farmed fish and prevalence, abundance and intensity data collected for comparison. Species determination within the genus Gyrodactylus is based upon subtle differences in hook morphology and has long posed a taxonomic problem. The discrimination of collected specimens was based on two platforms. The initial approach used classical morphometrics from the light microscope, the results being processed using multivariate analyses to separate species. The second approach analysed morphometric data collected from scanning electron micrographs. This was made possible by the development of a sclerite release technique utilising a source of ultrasound to liberate hooks from surrounding tissue and a subsequent flotation stage which permitted flat preparations. Sonication of fresh and frozen material retained the structures that would be lost by enzymatic digestion. The description of new morphometric parameters using digital image analysis allowed the subtle differences in hamuli and marginal hook shape to be discriminated when analysed using principal components analysis (PCA). Four species were identified following multivariate and morphological analyses of opisthaptoral sclerites. G. truttae Glaser, 1975 was found to occur on S. trutta and G. derjav;ni Mikailov, 1975 was found to occur on S. trllfta, S. salar and O. mykiss. Two hitheno undescribed forms, one on S. salar and one on S. alpinus which may be a new species are desclibed. In addition, two forms of G. derjavilli from S. salar and S. alpinlls and one form of G. truttae from S. tnltta are described. Sub-populations of Gyrodactylus sp. were found to be determined by the pattern of distribution of the host; S. salar. The two sub-populations were divided into a southern celtic population (Morph 1) and a nonhern boreal population (Morph 2). Water temperature, was found to be an important environmental parameter influencing sclerite size. The principal component analyses identified key characters which could discriminate G. salaris from the native British species using novel parameters based upon both single elements and the full complement of sclerites. Of these new parameters, the hamulus angle and the size of the marginal hook sickle aperture were the most discriminating. Electronmicrographs of hamuli were traced using a digitising tablet and prepared for image processing. The hamulus angle was measured on original hook images and on enhanced (skeletonised) images using an image analyser. Skeletonisation investigated the reliability of the hook angle as a taxonomic criterion by the removal of possible age-related sclerotisation of the hamulus. Statistical analysis of the data revealed that there are significant differences in hook angle between some species. The isolation of sclerites by sonication enabled their elemental composition to be investigated. The hamuli and marginal hooks were found to have a high sulphur content, indicative of a keratin-like substance. The ventral bar composed of sulphur and calcium is weakly keratinised. The hamulus and the ventral bar were also found to contain vanadium. the significance of which is unknown. The detailed morphology and composition of the individual sclerites is discussed in relation to the functional mechanics of the entire haptoral complex. The protein profiles of G. salaris. G. truttae and G. derjvani were investigated using SDS-PAGE gel electrophoresis, with four proteins common to the three species. Two antibodies raised against G. salaris were found using Western blots. The chaetotaxy of argentophilic structures on three species of the genus Gyrodactyllis was investigated to ascertain the usefulness of this technique in distinguishing species of this genus. Chaetotaxy maps were prepared for G. salaris from Scandinavia and compared to native species of Gyrodactylus parasitizing salmonids in Britain. A formula for the arrangement of the sensilla analogues and the evolutionary position of the genus Gyrodactylus is commented upon.

The importance of fish culture has been increasing since 1990’s. The steady growth of fish culture helps to ensure a stable supply of fish for human consumption. However, when compared with capture fisheries, production from fish culture is greatly influenced by fish diseases. Outbreaks of fish diseases have caused great economic loss to fish culture. Research has been conducted to understand and reduce the occurrence of fish diseases in fish culture. In Hong Kong, bacterial infection is the most common cause of fish diseases. This project is therefore directed to isolate and identify the causative bacterial pathogen of some fish disease cases with the aim of setting up a local fish disease database for assisting the identification of diseases and improving the understanding of fish diseases in fish farms in Hong Kong. In this project, seven fish disease cases caused by bacteria were investigated with the AFCD officials in Hong Kong. Nine fish disease bacterial pathogens were isolated and identified using different methods (including commercial biochemical test kits, automated system and DNA sequencing). The bacteria identified included Aeromonas hydrophila, Lactococcus garvieae, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus iniae, Vibrio vulnificus and Aeromonas salmonicida. Sensitivity tests to 10 common antibiotics conducted for the identified bacteria showed that spectinomycin is the most broad spectrum antibiotics. In addition, a long-term physical storage of bacterial stock with glycerol and glass beads was established for further research of the identified bacteria. For efficient data analysis, an electronic database using Microsoft Access to hold the identification results and case history of each isolated bacteria was developed. Different data entry forms and reports were also constructed to facilitate easy data entry and data access for users. The three bacteria identification methods were compared for their efficiency and accuracy. Some limitations encountered in this project including time constraints and low accuracy of some biochemical identification tests were discussed and recommendations to overcome these limitations and improvements to the constructed database were made.
published_or_final_version
Environmental Management
Master
Master of Science in Environmental Management

Isolates of Yersinia ruckeri were obtained from Europe, North America, Australia and South Africa. The biochemical and serological characteristics of the isolates were investigated. Biochemically the isolates were extremely uniform although motile, Tween positive isolates could be differentiated from non-motile, Tween negative isolates; these were designated biotypes 1 and 2 respectively. With the exception of two isolates, biotype 2 isolates were confined to the U. K. Five 0-serotypes were recognised and an O-serotyping scheme is proposed; the relation of this scheme to previously described schemes is discussed. The geographic distribution of the different serotypes is also discussed. The lipopolysaccharide (LPS) and outer membrane protein (OMP) profiles of isolates were analysed by SDS-PAGE and Western-blotting using both rabbit and rainbow trout antisera. The relation of LPS-type to 0- serotype, as well as variation within LPS-types, is discussed. Based on interstrain variation in the molecular weight of a heat-modifiable protein and of peptidoglycan-associated (porin) proteins, an OMP-typing scheme was developed. Three major OMP-types comprised 95% of the isolates studied. Variation in biotype, serotype and OMP-type was used as an epizootiological tool, and six serotype 01 clonal groups were recognised which differed in their geographic distribution. The production of iron-regulated OMPs and siderophores was investigated. Four iron-regulated OMPs were produced in all of the isolates examined; siderophores appeared not to be produced by any of the isolates. Production of iron-regulated OMPs was not an important virulence determinant and appears to be a chromosomally-mediated factor. Resistance to the bactericidal effects of normal rainbow trout serum and virulence were also investigated. Serum-resistance was associated principally with two serotype 01 clonal groups and virulence was associated with the same two clonal groups. Other serotype 01 clonal groups and other serotypes were iii generally serum-sensitive and avirulent. Thus, serum-resistance is an important virulence determinant in this organism. The role of outer membrane components in serum-resistance and virulence is discussed.

Rainbow trout macrophages were stimulated with PMA to produce 02- and H2O2 as detected by the reduction of nitroblue tetrazolium (NBT) and the oxidation of phenol red respectively. Addition of DDC or nitroprusside, inhibitors of superoxide dismutase (SOD) increased O2-levels and decreased H2O2 levels, whereas addition of exogenous SOD had the reverse effect. Such data are indicative of a respiratory burst pathway in teleost macrophages comparable with that of mammals. Respiratory burst activity, acid phosphatase activity and RNA synthesis in rainbow trout macrophages which have been stimulated in vitro with the mitogen Concanavalin A (Con A) or in vivo by injection of formalin-fixed Aeromonas salmonicida in Freund's incomplete adjuvant (FIA) was analysed. With Con A, in vitro stimulated head kidney (HK) or elicited macrophages had increased O2-production and RNA synthesis but no significant increases in H2O2 or acid phosphatase activity after 72h post-stimulation with Con A. In contrast, all functions were increased in in vivo stimulated macrophages compared with FIA-elicited peritoneal macrophages. In a bactericidal assay, Con A stimulated macrophages did not show an increase in killing of an avirulent strain of A. salmonicida (004) above control levels whereas in vivo stimulated macrophages not only displayed increased killing of the avirulent strain of bacteria but also acquired the ability to kill a virulent strain (048). Thus, Con A stimulated macrophages only possessed some of the features of activation whereas in vivo stimulated macrophages were activated as defined by the increased bactericidal activity. Peritoneal washes obtained in the collection of activated macrophages were able to increase NBT reduction in normal HK macrophages suggesting the presence of a soluble activating factor. Lymphokine (LK)-containing supernatants produced using either HK or blood derived leucocytes, by pulsing with 10ug/ml Con and 5ng/ml PMA, were able to increase O2- and H2O2 production, to enhance the killing of an avirulent strain of A. Salmonicida and conferred the ability to kill a virulent strain of A. salmonicida. The LK present in these supernatants was therefore designated a macrophage activating factor (MAF). The use of potential second signals to enhance the killing of bacteria by LK-treated macrophages, met with limited success. Only A. salmonicida (strain 004) LPS was able to produce a small increase in killing above LK-treatment. The MAF produced in this study was tested for antiviral/interferon (IFN) activity. The results showed that the supernatants did contain IFN activity. Attempts to semi-purify the MAF from antiviral activity showed the two activities to co-purify, indicating that both activities may be due to the same molecular species. The retention time of the MAF/IFN, coupled with the results of SDS-PAGE analysis showed the molecular weight of the moiety to be approximately 19K daltons. Both activities were sensitive to low pH (pH 2), high temperature (60oC) and trypsin, providing further evidence that the MAF and IFN activity produced in these studies may be due to the same molecular species, possibly akin to IFN- of higher vertebrates.

Proliferative Kidney Disease (PKD) caused by the Malacosporean parasite Tetracapsuloides bryosalmonae, is presently the most economically damaging disease of British rainbow trout farming, costing the industry in excess of £2.5 million per annum in the UK alone. With no vaccine or prophylactic treatment available, and only management techniques currently adopted to minimise the stress and mortality associated with the disease, alternative approaches must now be considered. This document investigates if selective breeding for PKD resistance is possible by assessing the level of additive genetic variation, and calculating the subsequent estimates of heritability, for commercial strains of rainbow trout. During a PKD outbreak on a commercial farm, 1500 communally reared juvenile rainbow trout from two strains (Houghton Spring and Isle of Man) were sampled on a single day, their body weight and fork length measured, and severity of kidney swelling scored according to the scale of Clifton-Hadley et al. (1987). Fish were assigned to individual families using microsatellite parentage assignment. Significant additive genetic variation was observed in the population, and families were ranked according to estimated breeding values. A combined estimate of heritability (h2 = 0.19 ± 0.08) for kidney score suggests the population will respond well to selective breeding for kidney score, which may be deemed a measure of resistance, whilst the favourable genetic correlations between kidney score and the production traits measured suggest simultaneous selection for kidney score and growth traits should also be effective. In order to support the findings of the initial research, controlled challenge experiments were conducted. Using the family EBV information on kidney score from the IoM strain (due to its certification as a disease-free site), four females, two with high and two with low response to PKD, were each crossed with a randomly selected neomale to produce twenty two families for PKD challenge experiments. The PKD experimental challenges showed evidence of additive genetic variation to kidney score over an eleven week period, supporting initial findings. A low score was deemed as evidence of greater resistance to the parasite in this study. Although female EBV was taken into consideration in the statistical model, there was found to be no significant difference in resistance according to family. Immunohistochemistry stained kidney sections from each individual involved in the challenges proved kidney score correlated significantly to the number of parasites in the kidney, suggesting that the scale of Clifton-Hadley et al. (1987) is a sufficient and accurate basis on which to describe the severity of PKD, and infection level in rainbow trout. Having discovered evidence that furunculosis, causative agent Aeromonas salmonicida, plays a major role in the mortality of fish suffering from PKD in the field, the bacterial disease was investigated to assess the resistance of the same families used in the PKD challenges. Twenty one of the families were used to discover that additive genetic variation for resistance to furunculosis is apparent when assessed as both a binary and longitudinal trait, suggesting significant genetic improvement can be made to increase resistance to furunculosis in the IoM stock. No significant correlation was observed between kidney score, EBV, and resistance to this bacterium, but there was a positive phenotypic correlation found between furunculosis resistance and size, suggesting simultaneous selection for performance and resistance is possible within this population.

The increase in aquaculture operations worldwide has provided new opportunities for the transmission of aquatic viruses. The occurrence of viral diseases remains a significant limiting factor in aquaculture production and for the sustainability. The ability to identify quickly the presence/absence of a pathogenic organism in fish would have significant advantages for the aquaculture systems. Several molecular methods have found successful application in fish pathology both for confirmatory diagnosis of overt diseases and for detection of asymptomatic infections. However, a lot of different variants occur among fish host species and virus strains and consequently specific methods need to be developed and optimized for each pathogen and often also for each host species. The first chapter of this PhD thesis presents a complete description of the major viruses that infect fish and provides a relevant information regarding the most common methods and emerging technologies for the molecular diagnosis of viral diseases of fish. The development and application of a real time PCR assay for the detection and quantification of lymphocystivirus was described in the second chapter. It showed to be highly sensitive, specific, reproducible and versatile for the detection and quantitation of lymphocystivirus. The use of this technique can find multiple application such as asymptomatic carrier detection or pathogenesis studies of different LCDV strains. The third chapter, a multiplex RT-PCR (mRT-PCR) assay was developed for the simultaneous detection of viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and sleeping disease (SD) in a single assay. This method was able to efficiently detect the viral RNA in tissue samples, showing the presence of single infections and co-infections in rainbow trout samples. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.

The increase in aquaculture operations worldwide has provided new opportunities for the transmission of aquatic viruses. The occurrence of viral diseases remains a significant limiting factor in aquaculture production and for the sustainability. The ability to identify quickly the presence/absence of a pathogenic organism in fish would have significant advantages for the aquaculture systems. Several molecular methods have found successful application in fish pathology both for confirmatory diagnosis of overt diseases and for detection of asymptomatic infections. However, a lot of different variants occur among fish host species and virus strains and consequently specific methods need to be developed and optimized for each pathogen and often also for each host species. The first chapter of this PhD thesis presents a complete description of the major viruses that infect fish and provides a relevant information regarding the most common methods and emerging technologies for the molecular diagnosis of viral diseases of fish. The development and application of a real time PCR assay for the detection and quantification of lymphocystivirus was described in the second chapter. It showed to be highly sensitive, specific, reproducible and versatile for the detection and quantitation of lymphocystivirus. The use of this technique can find multiple application such as asymptomatic carrier detection or pathogenesis studies of different LCDV strains. The third chapter, a multiplex RT-PCR (mRT-PCR) assay was developed for the simultaneous detection of viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and sleeping disease (SD) in a single assay. This method was able to efficiently detect the viral RNA in tissue samples, showing the presence of single infections and co-infections in rainbow trout samples. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.

The use of wrasse (Pisces: Labridae) as cleaner fish to combat infections with the parasitic copepods Lepeophtheirus salmonis (Kroyer) and Caligus elongatus (Nordmann) (sea-lice) in the culture of Salmo salar L. (Atlantic salmon) is now common. Infections with these parasites has caused considerable losses in the industry since its formative years. The use of the wrasse species Ctenolabrus rupestris (L. ) (goldsinny), Centrolabrus exoletus (L. ) (rockcook), Symphodus melops (= Crenilabrus melops) (L. ) (corkwing) and Labrus mixtus L. (cuckoo) as cleaner fish was first suggested in 1988. The use of these species in the industry is now widespread in Scotland, Ireland and Norway. The fish used are normally caught from the wild before being stocked with S. salar smolts during their first year at sea. The fish are routinely collected from waters close to the farm sites to be stocked. As most of the S. salar sea production sites in Scotland are located on the west coast of the country, the wrasse to be used in these sites are normally collected from these waters. The movement of wild fish into farm pens presents a risk of disease transfer from wrasse to S. salar and vice versa. Prior to their use as cleaner fish, these four species of wrasse had received little attention as subjects of scientific study. As a result, there was very little information available in the literature regarding their diseases. The present study was undertaken to investigate the potential pathogens present in wild populations in Scottish coastal waters, and, in particular, which of these pathogens, if any, could be transmitted to the S. salar. The study also investigated the susceptibility of wrasse to the two major viral diseases of S. salar to which they would be exposed in pens. In order to fully assess the pathogenicity of the potential disease agents under farm conditions, it was first necessary to establish the normal morphology of the wrasse species. Hence, a study of the morphological features of wrasse, with particular emphasis on those features important in the health of the fish was undertaken. Wrasse were shown to differ in many aspects from salmonids but shared many morphological features with other perciforme fish. Major differences from salmonids were evident in the skin, fins, pancreas, intestine, gonads and heart. There were also aspects of their morphology which differed from other perciforme fish, notably the structure of the heart. These features were regarded to be adaptations to the specific demands of their feeding strategies and habitats. This study was the first of its kind undertaken for wrasse and showed some early contraindications for the use of wrasse in culture; most notable was the marked lipid accumulation in, and resultant degeneration of, the liver resulting from the consumption of high energy S. salar feeds. Once the normal morphological features were established, it was possible to examine the disease status of wrasse. Wild fish were sampled from three different locations on the west coast of Scotland. These sites were all geographically distinct and were all used as sources of wrasse for the S. salar farming industry. Samples of wrasse were also obtained from farm sites supplied with wrasse from these wild sites, and an additional number of other geographically distinct farm locations. As a comparison wrasse were also obtained from a wrasse captive breeding facility and another captive location unrelated to the S. salar industry, a public aquarium. The fish from all of these sampling sites were examined fully for the presence of parasites, bacteria and, in some cases, viruses. Histological examination was also carried out on all of the fish studied. A total of 24 new parasite host records, and two tentative ones, were recorded from the four wrasse species studied. These new parasite records included protozoa, digeneans, nematodes and crustacea. Parasite infections were found to vary in prevalence, abundance and intensity in respect to the geographical characteristics of sampling sites and also the length of time spent in S. salar pens. It was concluded that the separation of wrasse from their natural diet and habitat influenced the degree of parasitism. None of the parasites found to infect wrasse were observed to cause any significant pathology in their hosts other than localised tissue responses. The possibility of transfer of wrasse parasites to S. salar was also investigated experimentally in a series of infections in which parasites dissected from wrasse were introduced to S. salar smolts by means of a novel gavage method. None of the parasites used established in the S. salar, indicating that there is little risk of transfaunation of parasites between wrasse and S. salar. However, this aspect requires further work due to the low number of parasites available and the subsequent low numbers of S. salar infected. Bacterial isolates were obtained from wrasse held in S. salar pens but were not found in any of the fish collected from the wild. Most of the bacterial strains isolated would normally be considered as opportunistic pathogens of fish. It was concluded that the relatively high levels of stress, both environmental and physical, that wrasse are subjected to under farm conditions were instrumental in the number of bacterial infections seen in wrasse. Only one pathogenic bacterial infection was seen in any of the fish sampled. This was an isolate of Aeromonas salmonicida, the agent known to cause the disease furunculosis, isolated from a wrasse obtained from one of the farm samples. Other authors have reported that this bacterium has already caused substantial losses of wrasse under farm conditions. It was concluded that Aeromonas salmonicida will prove to be a major pathogen of wrasse held in S. salar pens. No viruses wereI isolated from any of the wrasse studied. The susceptibility of wrasse to the most significant pathogens of S. salar under farm conditions was also subjected to investigation. In addition to sea-lice infection, the industry lists Infectious Pancreatic Necrosis (IPN) and Pancreas Disease (PD) as of primary importance for further research. Both of these diseases cause substantial losses in the industry. The susceptibility of wrasse to both of these disease conditions was investigated by means of experimental infections. In the case of IPN wrasse were infected by bathing with two different infective doses, a low dose which would be expected to induce the disease in S. salar parr and a second dose substantially higher than the first. The C. rupestris used were found to be susceptible to IPN. The wrasse developed some of the pathological characteristics typical of the disease in S. salar, however, other pathological signs were peculiar to wrasse. The recovery rate from the disease seen in wrasse was far more rapid than that recorded from S. salar. Shedding of the virus in the faeces of infected C. rupestris was also demonstrated. This study has illustrated for the first time the susceptibility of wrasse to IPN and that they can shed the virus in their faeces. This suggests that infected wrasse could be a source of continual reinfection in an affected sea site. Experimental infections of C. rupestris with PD followed a standard protocol for the reproduction of the disease in S. salar. Infection was by means of intraperitoneal injection with putatively infective material obtained from S. salar affected with PD. Two infection doses were used, the lowest dose used had been proven to be effective in inducing the disease in S. salar parr while the second dose, ten times higher than the first, had been shown to be effective in reproducing PD in S. salar smolts. The C. rupestris infected did not develop any of the typical signs of the disease seen in S. salar. It was, therefore, concluded that wrasse were not susceptible to PD.

Diseases which affect the central nervous system present a huge burden to sufferers and caregivers. In tandem with longevity, prevalences of age-related neurodegenerative diseases are increasing. However, despite the evident necessity for pharmaceutical interventions, there has been a distinct lack of drug development to combat these disorders. This is largely attributed to high financial costs of using rodent models. Thus the validation of a more cost-effective in vivo system would facilitate pharmaceutical screening. The work presented in this thesis addresses this issue by assessing the utility of zebra fish in two costly areas of translational neurobiology { lead identi cation and safety pharmacology. An aversive classical conditioning assay was developed and automated as a behavioural screening method. This robust assay allows fast assessment of cognition and cognitive decline. The effect of neurotoxin treatment on aversive learning was then assessed using this assay, demonstrating its efficacy as a screening tool for neurodegeneration research. Subsequently, a transgenic zebra fish line - expressing a mutated form of the Alzheimer's-associated human amyloid precursor protein - was assessed, demonstrating an age-related cognitive impairment. Additionally new genetic zebra fish lines were generated, which over-express genes (both endogenous and transgenic) related to Alzheimer's-like pathologies. Whilst these were not assessed within this thesis, they present promising tools for possible future investigations. Regarding safety pharmacology, regulatory bodies require all CNS-penetrant drugs be assessed for abuse potential. Zebra fish display reward responses to several common drugs of abuse (e.g. amphetamine, cocaine, morphine). Thus, the latter sections of this thesis evaluated the utility of zebra fish for assessing human abuse potential. A CPP paradigm was utilised to test a range of drugs, with the sensitivity and specificity of zebra fish compared to previous reports using rodent. Additionally, the development of a zebra fish drug discrimination assay was attempted. However the paradigms utilised failed to develop an efficacious assay.

The timing of seasonal events in salmonids is thought to be controlled by endogenous circannual rhythm(s) which are entrained by the seasonally-changing daylength. This thesis investigates the role of the pineal gland in the perception of the photoperiodic zeitgeber and the subsequent transmission of this information to the brain through neural or hormonal pathways. Melatonin biosynthesis by isolated rainbow trout pineal glands was shown to exhibit a differential response to graded photic or thermal stimuli. In vitro experiments were carried out at 10±0.50 C as this provided optimum melatonin levels for radioimmunoassay analysis together with a pineal longevity of up to 14 days. By incorporating a variety of light intensities into the light/dark cycle, the salmonid pineal gland was shown to synthesise significantly different levels of melatonin even when light levels varied by only 0.5 lux. Early work on the salmonid pineal suggested it was unresponsive to red light, having a spectral sensitivity which peaks between 500 and 550 nm, this study has revealed that the pineal is also capable of responding to wavelengths between 660 to 800 nm, at which pineal reception was previously thought to be severely limited. No endogenous rhythm of melatonin secretion was observed within the isolated rainbow' trout pineal gland. Both Atlantic salmon and Atlantic halibut pineals exhibited elevated levels- of melatonin in response to the dark phase, however, they also appeared capable of maintaining this rhythm in the absence of external stimuli. This provides the first evidence that the daily rhythm of melatonin production in these species is controlled by an endogenous circadian oscillator located within the pineal II gland. The pinealectomy technique developed during the course of this thesis successfully abolished the diel rhythm of melatonin secretion and, together with an enucleation procedure, enabled the pineal to be identified as the predominant source of the dark phase melatonin in Atlantic salmon and rainbow trout. However, the lateral eyes did contribute significantly to plasma melatonin levels in both species. Long term experiments, involving pinealectomy and/or implantation of melatonin, were used to investigate the role of the pineal gland in the timing of rainbow trout maturation and smoltification in Atlantic salmon. Pineal removal at the summer or winter solstices did not significantly alter the timing of smoltification. However, significantly higher blood serum osmolarities following seawater challenge tests were observed in smolts implanted with melatonin. This, together with a significant growth increase shown by salmon parr within 1 month of implantation, indicates that melatonin may directly affect the development of salmonids through either a physiological response or by influencing the entrainment of endogenous rhythms. The increased growth observed in the implanted parr is also thought to be responsible for the unimodal population distribution and high percentage of S1 smolts within this group. Investigations into the role of the pineal gland in the timing of spawning in rainbow trout found that pineal removal at the summer solstice caused a 6 week delay in spawning time compared to intact fish. However, no clear effects on spawning time were observed when pineal removal, with or without melatonin implantation, was performed to coincide with the change from long to short daylengths which is known to advance spawning times. Although no significant effect in spawning times was observed between groups, the 4 month spawning period of the pinealectomised group compared to 1 month in the shampinealectomised fish also suggested that pineal removal may have caused a desynchronisation in spawning time. Pinealectomy and/or implantation did not alter egg size or fecundity, but plasma calcium levels were shown to be significantly lower in the pinealectomised trout over the spawning period. To summarise, the pineal gland and melatonin play a significant role in salmonid development. It is suggested that melatonin can influence biological systems through a direct physiological action while the pineal gland may synchronise circannual events through the photoneuroendocrine transduction of seasonal environmental information.

Une partie de ce travail de thèse a consisté à appliquer les méthodes de FISH pour l’étude de trois bactéries pathogènes intracellulaires. La viabilité de Bartonella henselae a été évaluée à partir de ganglions de patients atteints de la maladie des griffes du chat (CSD). Le faible taux d’ARN détecté par biologie moléculaire, la stérilité des cultures, l'absence de détection par analyses histologiques et FISH confirment que B. henselae n'est pas ou rarement viable dans les ganglions de patients atteints de CSD. Tropheryma whipplei, l’agent de la maladie de Whipple, a été identifié et localisé par FISH, dans les macrophages d’un ganglion et d’une biopsie pulmonaire, confirmant le diagnostic infectieux. Deux méthodes de FISH ont été testées pour détecter Coxiella burnetii dans des cas d’endocardites et d’infections vasculaires en utilisant des sondes oligonucléotidiques et des sondes PNA. Les résultats ont confirmé une meilleure efficacité des sondes PNA et démontré que les techniques de FISH sont plus sensibles que l’immunohistochimie pour le diagnostic des endocardites et des infections vasculaires à C. burnetii. Nous avons également évalué les stratégies moléculaires mises en place pour le diagnostic syndromique. Bien que la PCR conventionnelle à large spectre permette l'identification de micro-organismes fastidieux et anaérobies, la PCR spécifique en temps réel révèle une supériorité significative dans le diagnostic syndromique. En conclusion, ce travail a permis de démontrer l’efficacité et l’applicabilité de la FISH pour la détection bactérienne. Cette méthode peut être utilisée comme un outil complémentaire afin d'améliorer le diagnostic de microbiologie clinique
We applied FISH methods to the study of three intracellular pathogenic bacteria. The viability of Bartonella henselae was evaluated in a large series of lymph nodes from patients with cat scratch disease (CSD). The results obtained, associated with sterile cultures and negative histological analyzes and FISH, as well as the low level of RNA detected by molecular biology, provide evidence that B. henselae are not or are rarely viable in the lymph nodes of patients with CSD. Tropheryma whipplei has been identified by FISH in macrophages from one lymph node and for the first time in a pulmonary biopsy, confirming the diagnosis of infection. Two methods of FISH have been tested to detect Coxiella burnetii in cases of endocarditis and vascular infections using oligonucleotide and PNA probes. The results attested to the greater efficiency of PNA probes, and demonstrated that FISH were applicable for the diagnosis of C. burnetii endocarditis. We also evaluated the molecular strategies used for syndrome-driven diagnosis of infectious diseases. Although conventional broad-spectrum PCR allows for the identification of fastidious and anaerobic microorganisms, real-time specific PCR reveals a significant superiority in syndrome-driven diagnosis. The addition of specific PCRs in real time PCR would improve our molecular strategies, for example, in the case of the detection of Staphylococcus aureus for the diagnosis of lymphadenopathy. In conclusion, this work demonstrates the effectiveness and applicability of FISH for the identification of intracellular bacteria. This method can be used as an important complementary tool to the improvement of clinical microbiological diagnosis

Streptococcus agalactiae infection is one of the major disease problems affecting farmed tilapia (Oreochromis niloticus) worldwide. Tilapia are highly susceptible to this disease which results in mortality of up to 70% over a period of around 7 days and significant economic losses for farmers. Affected tilapia commonly present with an irregular behaviour associated with meningoencephalitis and septicaemia. Currently, factors affecting the virulence and transmission of S. agalactiae in fish including tilapia are poorly understood. Reports from natural outbreaks of S. agalactiae infection on tilapia farms have suggested larvae and juvenile or fish smaller than 20 g are not susceptible. In addition, there is variability in individual response to experimental inflammatory challenge associated with coping styles (bold, shy) in common carp (Cyprinus carpio). The central hypotheses of this thesis were that weight, age and coping style might affect the development and progression of this bacterial disease. This study investigated these three factors with experimental S. agalactiae infection in Nile tilapia. A range of bacterial isolates recovered from farmed tilapia, presenting with clinical sign of streptococcosis during natural disease outbreaks were identified and characterised as S. agalactiae by standard conventional methods, biochemical characteristic tests, Lancefield serogrouping and species-specific PCR assay. These isolates were Gram-positive cocci, either β- or non-haemolytic (γ), non-motile, oxidase negative and all of serogroup B. In addition, they were able to grow on Edwards medium (modified) agar as blue colonies and growth was observed in broth from 22 to 37 oC and with 0.5-5% NaCl. The biochemical profiles showed some differences in reactions while all the PCR samples showed similarities to the S. agalactiae type strain. These data confirmed that these strains were identified as group B S. agalactiae. A challenge model for S. agalactiae in Nile tilapia was developed and the LD50 estimated prior to performing subsequent experimental challenge studies. Two exposure routes, immersion and intraperitoneal injection (i.p.), were tested with various concentrations of S. agalactiae. Only i.p. injection produced significant mortalities (9 × 108 CFU/ml = 48% mortality, 9 × 107 = 48% and 8 × 106 = 26%). Streptococcus agalactiae was recovered and identified from all the dead and moribund fish during these experiments, where affected fish showed similar clinical signs and pathology to those reported from natural S. agalactiae infections. The study results showed that an experimental i.p. challenge model for S. agalactiae infection had successfully infected healthy Nile tilapia. In the immersion challenges, only 1 fish died despite testing a range of bacterial concentrations, exposure times, stocking density, water system and bacterial preparations. The experimental studies were conducted to investigate the association between weight or age of fish and susceptibility to S. agalactiae infection in Nile tilapia. This was performed under experimental conditions including control groups and a single population of 8 months old fish from one set of parents divided into 7 weight categories. These fish received a single i.p. injection of 6 × 107 CFU/ml of S. agalactiae. Controls and fish of 4 or 8 months old with a mean weight of 5 g received an i.p. injection of 7 × 107 CFU/ml of S. agalactiae. Clinical signs, lesions and histopathological changes in the affected fish were consistent with those reported in natural infection. Streptococcus agalactiae was recovered and identified from all moribund or dead fish. The mortality in the study of different weights varied from 0 to 33% between the groups but the association with weight was weak (R2 = 0.02). In the study of different ages the 4 months old fish group had a total mortality of 24%, and the 8 months old fish group a total mortality of 4%. This study produced no evidence for an association between the weight and susceptibility to S. agalactiae infection but suggested an association between the age or growth rate of fish and this disease. Different coping styles and susceptibility to S. agalactiae infection in Nile tilapia was examined. Fish were screened and scored depending on their risk-taking behavioural responses to a range of different environmental conditions. Individual differences in behavioural responses were evident but only consistent across behavioural trials for some individuals. A selection of fish with consistent responses across trials was exposed to the 6 × 107 CFU/ml of S. agalactiae by i.p. injection. Fewer bold than shy fish died suggesting that the bold fish might be less susceptible to the infection than shy fish. In conclusion, this study characterised a number of S. agalactiae isolates and developed an experimental bacterial challenge model. Subsequent experiments suggested that age (or growth rate) and coping style in fish but not the fish weight may affect susceptibility to S. agalactiae infection in Nile tilapia.

Piscine francisellosis, caused by Francisella noatuenensis subsp orientalis (Fno), is an emerging infectious disease in the tilapia industry, but no effective commercial treatments or vaccines are available. The use of immunostimulants is a promising method to control diseases in aquaculture, and various algae and algal-derived compounds are potent immunostimulants for improving immune status. Algae produce a great variety of secondary metabolites that exert a broad spectrum of biological activities. The aim of this thesis was to evaluate the effectiveness of algal compounds against Fno in vitro and in vivo and determine their potential to control francisellosis infection in Nile tilapia Oreochromis niloticus L. under experimental conditions, and in an alternative host, namely the greater wax moth Galeria mellonella. Some of the algae and their compounds (Chlorella sp., alginic acid, and ß-glucan) exerted antimicrobial activity in vitro against Fno, Aeromonas hydrophila and Streptococcus agalactiae and stimulated responses of Nile tilapia macrophages (Chapter 2). An immersion challenge model for Fno STIR-GUS-F2f7 was developed in two genetic groups of Nile tilapia, and the homo gold strain was more susceptible to infection than wild type (Chapter 3). In vivo trials were conducted in Nile tilapia homo gold where fish were fed diets supplemented with 10% Scenedesmus quaricauda, 10% Haematococcus pluvialis, and 0.1% or 0.2% alginic acid or ß-glucan, and then challenged with Fno and co-infected with S. agalactiae (Chapter 4). The Fno challenge failed to produce mortality; however, co-infection resulted in high mortalities in all groups. As the in vivo trial in tilapia could not be to repeated, a G. mellonella model for Fno was validated. Fno doses between 0.7–1.7 x 108 CFU mL-1 killed G. mellonella, while tetracycline, alginic acid and ß-glucan rescued the wax moth from lethal doses of bacteria (Chapter 5).

Pacific salmon reared commercially off of the Coast of British Columbia suffer great mortality losses to Bacterial Kidney Disease (BKD), caused by the diplobacillus bacterium Renibacterium salmqninarum. This thesis investigates the effects of environmental conditions on the growth performance and disease susceptibility of salmonids reared in captivity. I found that growth rate of chinook salmon was significantly higher in fish fed to 100 compared to 67 % of satiation during the first 175 days of saltwater rearing but not during the first winter. Feed coversion rate was significantly higher for fish fed at 100 % of satiation compared to 67 % of satiation and higher during the winter compared to summer and fall, irrespective of feeding level. Mortality rates were significantly higher during the summer than during the fall or winter, irrespective of experimental treatment. The last BKD sampling period (day 263) revealed that infection rates were directly proportional to stocking densities of 1.5 to 4 kg.m⁻₃. Hatchery-reared chinook salmon held in freshwater aquaria had significantly lower hematocrit and plasma cortisol concentration increases in response to increased stocking density than did their wild counterparts. Crowding of hatchery-reared and wild chinook salmon resulted in equally increased mortality rates for both groups of fish. Day 33 plasma cortisol concentrations in Atlantic salmon held at three stocking densities were directly proportional to stocking densities of 8 to 64 kg.m⁻₃. The ability of anterior kidney lymphocytes from these fish to produce antibody-producing cells was inversely proportional to the density at which the fish were held.
Land and Food Systems, Faculty of
Graduate

The expanding aquaculture industry continues to encounter major challenges in the form of highly contagious aquatic viruses. Control and eradication measures targeting the most lethal and economically damaging virus-induced diseases, some of which are notifiable, currently involve ‘stamping out’ policies and surveillance strategies. These approaches to disease control are performed through mass-culling followed by restriction in the movement of fish and fish products, resulting in considerable impacts on trade. Although effective, these expensive, ethically complex measures threaten the sustainability and reputation of the aquatic food sector, and could possibly be reduced by emulating innovative vaccination strategies that have proved pivotal in maintaining the success of the terrestrial livestock industry. DIVA ‘differentiating infected from vaccinated animal’ strategies provide a basis to vaccinate and contain disease outbreaks without compromising ‘disease-free’ status, as antibodies induced specifically to infection can be distinguished from those induced in vaccinated animals. Various approaches were carried out in this study to assess the feasibility of marker/DIVA vaccination for two of the most important disease threats to the global Atlantic salmon and common carp/koi industries, i.e. infectious salmon anaemia (ISA) and koi herpesvirus disease (KHVD), respectively. Antibody responses of Atlantic salmon (Salmo salar L.), following immunisation with an ISA vaccine, administered with foreign immunogenic marker antigens (tetanus toxoid (TT), fluorescein isothiocyanate (FITC) and keyhole limpet hemocyanin (KLH)) were assessed by antigen-specific enzyme linked immunosorbent assay (ELISA). Although antibodies were induced to some markers, these were unreliable and may have been affected by temperature and smoltification. Detectable antibodies to ISAV antigen were also largely inconsistent despite low serum dilutions of 1/20 being employed for serological analysis. The poor antibody responses of salmon to the inactivated ISA vaccine suggested that DIVA vaccination is not feasible for ISA. A similar approach for KHV, utilising green fluorescent protein (GFP) as the marker, similarly failed to induce sufficiently detectable antibody responses in vaccinated carp (Cyprinus carpio L.). However, as high anti-KHV antibody titres were obtained with an inactivated KHV vaccine (≥1/3200), alternative approaches were carried out to assess the feasibility of DIVA vaccination for carp. Investigations of early KHV pathogenesis in vivo and antigen expression kinetics in vitro (0-10 days post infection (dpi)) provided valuable data for the diagnostics necessary for DIVA surveillance strategies. Following viral infection, molecular methods were shown to be the most effective approach for early detection of KHV infected fish prior to sero-conversion, during which time antibodies are not detectable. An experimental immersion challenge with KHV, however, revealed complications in molecular detection during early infection. The KHV DNA was detected in external biopsies of skin and gills, but also internally in gut and peripheral blood leukocytes ≤ 6 hours post infection (hpi), suggesting rapid virus uptake by the host. The gills and gut appeared to be possible portals of entry, supported by detection of DNA in cells by in situ hybridisation (ISH). However, many false negative results using organ biopsies occurred during the first 4 dpi. The gills were the most reliable lethal biopsy for KHV detection by various polymerase chain reaction (PCR) assays, with a PCR targeting a glycoprotein-gene (ORF56) and a real-time PCR assay being the most sensitive of the 7 methods investigated. Importantly, non-lethal mucus samples reduced the number of false negative results obtained by all KHV PCR assays during the earliest infection stages with large levels of viral DNA being detected in mucus (up to 80,000 KHV DNA genomic equivalents 200 μL-1). KHV DNA was consistently detected in the mucus as a consequence of virus being shed from the skin. Determining the expression kinetics of different viral structural proteins can be useful for DIVA serological tests. Analysis of KHV antigen expression in tissues by immunohistochemistry and indirect fluorescent antibody test was inconclusive, therefore 2 novel semi-quantitative immunofluorescence techniques were developed for determining KHV antigen expression kinetics in susceptible cell lines. During the course of KHV infection in vitro, a greater abundance of capsid antigen was produced in infected cells compared to a glycoprotein antigen (ORF56), as determined by detection with antigen-specific monoclonal antibodies (MAbs). The capsid antigen was characterised as a ~100 kDa protein by SDS-PAGE and identified as a product of KHV ORF84 by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF/TOF MS). This antigen was subsequently detected in the serum of >25% of KHV infected/exposed carp (6/17), as well as in carp vaccinated with a live attenuated vaccine (3/4), but not with an inactivated vaccine (0/7), by Western blot making it a potential DIVA target for an inactivated vaccine. Attempts were made to improve the sensitivity of KHV serological testing by taking advantage of recombinant proteins specific for KHV (CyHV-3), rORF62 and rORF68 and eliminating any interference by cross-reacting antibodies to carp pox (CyHV-1). These proteins successfully reacted with anti-KHV antibodies. The feasibility of DIVA strategies for KHVD was determined using these recombinant antigens to coat ELISA plates. Differential antibody responses were detected from carp sera to an internal virus tegument protein (rORF62) and external region of a transmembrane protein (rORF68). Fish vaccinated with an inactivated vaccine produced significantly lower antibody responses to rORF62 than to rORF68, whereas infected, exposed and live attenuated vaccinated fish recognised both proteins allowing differentiation between vaccinated and infected carp. However, the sensitivity of the assay was limited, possibly by high levels of natural antibodies detected at the relatively low serum dilutions (1/200) used. As the capsid antigen (ORF84) and tegument protein (ORF62) are derived from internal KHV structural proteins, they induce non-neutralising antibodies, which may be useful for DIVA strategies. Such antibodies are longer lasting than neutralising antibodies and often comprise the majority of fish anti-viral antibodies. This was noted in a fish surviving experimental challenge, which had an antibody titre of 1/10,000, but neutralising titre of 1/45. Such antigens may therefore hold potential for developing effective serological diagnostic tests for KHV and provide the potential for DIVA strategies against KHVD. Natural antibodies will, however, continue to present a challenge to the development of sensitive and reliable KHV serological tests, and hence the application of DIVA strategies.

Rainbow trout represents the most prominent species in freshwater farming in UK aquaculture. One of the common diseases constraining rainbow trout production and increasingly causing problems in Atlantic salmon (Salmo salar L.) hatcheries worldwide is rainbow trout fry syndrome (RTFS) or bacterial cold water disease (BCWD). During the last 20 years, the development of a commercial vaccine against RTFS has been hindered by the prevalence of a wide range of the fish pathogen F. psychrophilum, thus the current treatment of choice is the use of antibiotics. Studies involved in understanding the innate and adaptive immune response of vaccinated rainbow trout fry using inactivated whole cell are still lacking. Therefore, the aim of this thesis is to characterise the strain diversity and antibiotic susceptibility of UK F. psychrophilum isolates, evaluate the efficacy of a whole-cell formalin-killed polyvalent vaccine, which was developed based on the characterisation results of this study, and investigate the immune response in trout fry following the immersion vaccination via the changes in expression of relevant immune genes. A total of 315 F. psychrophilum isolates, 293 of which were collected within the UK, were characterised using four genotyping methods and a serotyping scheme. A high strain diversity was identified among the isolates with 54 pulsotypes, ten (GTG)5-PCR types, two 16S rRNA allele lineages, seven plasmid profiles and three serotypes. The predominant profile observed within the F. psychrophilum isolates examined was PFGE cluster II – (GTG)5-PCR type r1 – 16S rRNA lineage II – serotype Th (n= 70/156, 45%). The characterisation results not only revealed the wide distribution within the UK and the persistence within a site of predominant pulsotypes, but also the presence of unique genotypes in certain sites or countries. Co-existence of genetically and serologically heterogeneous isolates within each farm was detected, highlighting the reasons this disease is so difficult to control, especially by vaccination. The occurrence over time of F. psychrophilum pulsotypes within a site could provide important epidemiological data for farm management and the development of site-specific vaccines. The antimicrobial susceptibilities of 140 F. psychrophilum strains, 125 of which were from the UK, were evaluated by the broth microdilution (MIC) and disc diffusion methods. There was evidence of reduced susceptibilities to three of the main antimicrobials used in UK aquaculture. Broth microdilution testing showed that only 12% of 118 UK isolates tested were WT to oxolinic acid (MIC COWT 0.25 mg L-1), 42% were WT for oxytetracycline (MIC COWT 0.25 mg L-1), and 66% were WT for amoxicillin. In contrast, all the isolates tested were WT (MIC COWT 2 mg L-1) for florfenicol, the antimicrobial of choice for RTFS control in the UK. Despite the imprecision of disc diffusion-based COWT values due to high standard deviations, there was a high categorical agreement between the classification of the strains (into WT or NWT) by MIC and disc diffusion methods for florfenicol (100%), oxolinic acid (99%), amoxicillin (97%) and oxytetracycline (94%). In general, this study showed that the UK F. psychrophilum isolates examined remain susceptible to florfenicol and also stresses the importance of performing susceptibility testing using standardised methods and COWT values. Several statistically significant associations between genotypes and the reduced susceptibilities of F. psychrophilum strains were revealed. A whole-cell formalin killed polyvalent vaccine against RTFS/BCWD was developed by combining three genetically and serologically divergent strains, recently collected from UK farms. The efficacy of this polyvalent vaccine was evaluated after immersion vaccination in 5 g trout and bath challenge using hydrogen peroxide as a pre-stressor with a virulent heterologous isolate of F. psychrophilum strain. Significant protection was achieved with an RPS of 84%. The combination of exposure to hydrogen peroxide prior to bath challenge may be an alternative to an injection challenge with 12 g trout, although further standardisation and optimisation of the challenge model is required. Changes in the innate immune response of trout fry following the initial vaccination included the up-regulation of the interleukin 1 β (IL-1β) gene in head kidney at 4 h and the up-regulation of toll-like receptor-2 (TLR-2) in skin at day 2. While the expression levels of C3 was unchanged, the down regulation of CD8-α in head kidney and spleen and CD4-1 in spleen were documented. IgM and IgT transcripts were found to be up-regulated in hind-gut two days post-vaccination. Understanding the strain diversity and the antibiotic susceptibility of UK F. psychrophilum isolates could help improve the control strategies, such as preventing the spreading of pathogenic F. psychrophilum clones between fish farms, reducing the use of antibiotics in RTFS/BCWD treatment and monitoring the development of acquired antibiotic resistance mechanisms. Moreover, strain characterisation data of UK F. psychrophilum species has assisted in selecting suitable candidates for developing an effective RTFS vaccine.

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.

Em virtude do exponencial crescimento dos cultivos comerciais do beijupirÃ, Rachycentron canadum, em paÃses da Ãsia, tais como China e Taiwan, com perspectivas promissoras tambÃm para o Brasil, fazem-se necessÃrias pesquisas que visem fornecer bases tecnolÃgicas para a sustentabilidade da atividade no Nordeste brasileiro, como o desenvolvimento de formulaÃÃes de raÃÃes eficientes. O presente estudo objetivou analisar a sanidade desses organismos submetidos à condiÃÃo experimental, com a finalidade de observar nÃveis aceitÃveis de farelo de soja em dietas balanceadas para essa espÃcie. Foram testadas cinco dietas experimentais formuladas para conter 48% de ProteÃna Bruta (PB) e 12% de Extrato EtÃreo (EE). As cinco dietas variaram no percentual de substituiÃÃo de farinha de peixe por farelo de soja, a partir da dieta base, em quatro nÃveis: 12% (CB12); 25% (CB25); 37% (CB37) e 50% (CB50). Avaliou-se tambÃm uma dieta comercial para peixes marinhos contendo 40% PB e 8% EE. As dietas foram ofertadas durante 56 dias. Na despesca foram amostrados cinco indivÃduos de cada tratamento, os quais foram analisados quanto à integridade das estruturas externas; analise a fresco do muco e brÃnquias; coleta de sangue e de tecidos para anÃlises em laboratÃrio. Na anÃlise a fresco foram observados protozoÃrios do gÃnero Epystilis e nematoides. Os resultados hematolÃgicos indicaram que elevadas inclusÃes de farelo de soja afetam o padrÃo dos leucÃcitos do beijupirÃ, elevando a contagem de neutrÃfilos e diminuindo a de linfÃcitos nas dietas CB37 e CB50. A histologia revelou maior infiltraÃÃo de leucÃcitos na lÃmina prÃpria do intestino distal na dieta CB37, bem como uma diminuiÃÃo das vacuolizaÃÃes supranucleares do tratamento CB50. Os resultados sugerem que nas condiÃÃes avaliadas, atà 25% de substituiÃÃo da farinha de peixe pelo farelo de soja nÃo impactam significativamente na sanidade do beijupirÃ, embora se tenha observado inÃcio de lesÃes neste tratamento, que se agravaram nas demais substituiÃÃes. A raÃÃo comercial impactou de forma semelhante Ãs piores dietas para os organismos.
Since the exponential growth on commercial culture of cobia, Rachycentron canadum, in Asian countries like China and Taiwan with good perspectives to Brazil, new insights of research such as the development of efficient feed formulations, have been needed to provide technological bases and sustainability of this sector on Brazilian Northeast. The present study aimed to analyze cobiaâs health under experimental condition and acceptable levels of soybean meal in balanced diets by this species. It was tested five experimental diets formulated to contain 48% of Crude Protein (CP) and 12% of Crude Fat (CF). It was changed the percentage content of fishmeal replacement by soybean meal, from basal diet, at four levels: 12% (CB12); 25% (CB25); 37% (CB37) e 50% (CB50). It was also evaluated a commercial feed to marine fishes with 40% PB e 8% EE. The diets were offered for a 56-days period. On the harvest, five fish were sampled from which treatment and observed for external structures integrity; mucus was analyzed in a wet mount; the blood and tissues collection to laboratorial assessment. It was observed Epystilis protozoan and nematodes in wet mount. Hematological results have showed that the higher inclusion levels of soybean meal level did affect the normal total counting numbers of leucocytes of cobia, increasing neutrophils and decreasing total counting numbers of lymphocytes CB37 and CB50 diets. The histology showed that most of leucocytes were migrating in distal intestine of lamina propria, diet CB37, as well as decreasing of supranuclear vacuolization in diet CB50. The results suggest that up to 25% crude protein in form of soybean meal has not had a significant impact in cobiaâs health. The commercial marine fish feed impacted similarly to worst diets.

Algumas doenças associadas à piscicultura de tilápias trazem enormes prejuízos aos criadores, resultando em expressivos coeficientes de morbidade e mortalidade entre animais de todas as idades. Nesse sentido, exigências para o aprimoramento do diagnóstico laboratorial de doenças de natureza infecto-contagiosa tornaram-se constantes, face às perdas vinculadas. Os Ranavirus são vírus que infectam vertebrados ectotérmicos causando necrose generalizada, hemorragias focais e apoptose celular nos animais infectados. O presente estudo objetivou a realização de infecções experimentais em larvas e alevinos de tilápia do Nilo (Oreochromis niloticus), através de 3 diferentes modelos de infecção, utilizando estirpe de Ranavirus Frog vírus 3-like; recentemente detectada e isolada no Brasil, para avaliação das patologias associadas, incluindo o sequenciamento e análise do genoma da cepa viral. Vários sinais clínicos macroscópicos e microscópicos foram observados, hemólise, hemácias com anisocitose e policromasia, alterações relacionadas a média dos volumes das hemácias e hemoglobina, índices hepato-somáticos e espleno-somáticos com interações estatisticamente significantes, linfócitos reativos, alterações nos níveis das enzimas alanina aminotransferase e corpúsculos de inclusão basofílicos em diferentes tecidos dos animais experimentados com Ranavirus FV3-like foram observados dentro do período de 60 dias pós-infecção. Além disso, animais experimentalmente infectados foram positivos ao Ranavirus através de qPCR. O genoma de cepa brasileira de Ranavirus FV3-like apresentou 105 kilobases, contendo 54,98% de guanina + citosina, sendo 94 potenciais ORFs anotadas. A reconstrução filogenética, baseada em sequencias nucleotídicas, agrupou a amostra brasileira de Ranavirus no clado dos Frog vírus 3-like e as maiores identidades do genoma se deram com cepas norte-americanas de FV3-like. Doze potenciais eventos de recombinação, estatisticamente significantes (p < 0,05), foram identificados entre a cepa brasileira e amostras de referência de Ranavirus FV3-like. Nesse sentido, o presente trabalho contribui para o melhor entendimento da infecção causada por estirpe brasileira de Ranavirus FV3-like em tilápias do Nilo (Oreochromis niloticus). Além disso, adiciona e reforça a potencial recombinação entre diferentes estirpes de Ranavirus, colaborando para uma melhor compreensão das características de agentes virais associados a graves surtos em vertebrados ectotérmicos no país.
Some diseases associated with tilapia fish farming cause enormous losses to breeders, resulting in significant morbidity and mortality rates among animals of all ages. In this sense, requirements for the improvement of the laboratory diagnosis of diseases of infectious-contagious nature, became constant, in view of the related losses. Ranaviruses are viruses that infect ectothermal vertebrates causing widespread necrosis, focal haemorrhages and cell apoptosis in infected animals. The present study aimed to perform experimental infections in Nile tilapia (Oreochromis niloticus) fry and larvae through 3 different infection models, using Ranavirus Frog virus 3-like strain recently detected and isolated in Brazil, to evaluate the associated pathologies, including sequencing and genome analysis of the viral strain. Several macroscopic and microscopic clinical signs were observed, hemolysis, red blood cells presenting anisocytosis and polychromasia, alterations related to the mean red blood cell volumes and hemoglobin present in the red blood cells, hepato-somatic and spleno-somatic indexes showing statistically significant interactions, reactive lymphocytes, alterations in the levels of the enzymes alanine aminotransferase and basophilic inclusion corpuscles in different tissues of animals tested with Ranavirus FV3-like were observed within the 60-day post-infection period. In addition, experimentally infected animals were positive to Ranavirus through qPCR. The genome of the Ranavirus FV3-like Brazilian strain presented 105 kilobases, containing 54.98% of guanine + cytosine, with 94 potential ORFs annotated. The phylogenetic reconstruction, based on nucleotide sequences, grouped the Brazilian sample of Ranavirus in the clade of Frog virus 3-like, the greater identities of the genome were with North American strains of FV3-like. Twelve potential recombination events, statistically significant (p <0.05), were identified between the Brazilian strain and reference samples of Ranavirus FV3-like. The present work contributes to a better understanding of the infection caused by the Brazilian strain of Ranavirus FV3-like in Nile tilapia (Oreochromis niloticus). In addition, it adds and reinforces the potential recombination between different strains of Ranavirus, contributing to a better understanding of the characteristics of viral agents associated with severe outbreaks in ectothermic vertebrates.

Práticas nutricionais saudáveis e a globalização cultural popularizaram o consumo de pratos à base de peixe cru, anteriormente restritos aos países orientais. Estimativas mostram que doenças de origem alimentar causam aproximadamente 76 milhões de casos, 325 mil hospitalizações e 5 mil mortes a cada ano, somente nos Estados Unidos. Casos com etiologia desconhecida somam 62 milhões, com 265 mil hospitalizações e 3.200 mortes. O objetivo deste estudo foi investigar a presença de Escherichia coli, Salmonella spp. e Staphylococcus aureus e cepas potencialmente patogênicas de Aeromonas spp. e Vibrio spp. em peixes comercializados em feiras livres da cidade de São Paulo, Brasil. Vinte amostras de peixes, de diferentes espécies, foram adquiridas em feiras livres e analisadas utilizando metodologia convencional para investigação de patógenos em alimentos. Altos níveis de contaminação fecal foram detectados em 25% das amostras. Staphylococcus aureus foi isolado em 10% das amostras, entretanto em valores abaixo do permitido pela legislação brasileira. Todas as amostras estavam negativas para Salmonella spp. V. parahaemolyticus não foi isolado, 30% das amostras foram positivas para outras espécies de Vibrio, inclusive Vibrio cholerae não-O1/não-O139. Aeromonas spp. , incluindo A. hydrophila foi isolada em 50% das amostras de peixe. O isolamento de Vibrio cholerae não-O1/não-O139 e Aeromonas hydrophila, assim como Staphylococcus aureus e Escherichia coli, sugere que peixes comercializados em feiras livres da cidade de São Paulo podem representar um risco para os consumidores e ser um importante veículo de transmissão de espécies enteropatogênicas.
Healthier nutritional lifestyles and cultural globalization have popularized the consumption of raw fish dishes that were previously restricted to oriental countries. Estimates indicate that food-borne diseases cause approximately 76 million illnesses, 325,000 hospitalizations and 5,000 deaths each year in the United States alone. Cases with unknown etiology account for 62 million illnesses, 265,000 hospitalizations and 3,200 deaths. The purpose of this study was to investigate the presence of Escherichia coli, Salmonella spp., Staphylococcus aureus and potentially pathogenic strains of Aeromonas spp. and Vibrio spp. in fish commercialized at the retail level in the markets of São Paulo City, Brazil. Twenty fish of different species were analysed for foodborne pathogens using conventional methodologies. High levels of faecal contamination were detected in 25% of fish samples. Staphylococcus aureus was isolated from 10% of samples; however, in each case this was below the limits established by Brazilian legislation. All samples were negative for Salmonella and 30% tested positive for others Vibrio spp., including Vibrio cholerae non-O1/non-O139. Vibrio parahaemolyticus was not found in this study. Aeromonas spp., including A. hydrophila, were isolated in 50% of fish samples. The isolation of Vibrio cholerae non-O1/non-O139 and Aeromonas hydrophila as well as Staphylococcus aureus and Escherichia coli suggests that fish commercialized in São Paulo City may represent a health risk to consumers and be an important vehicle for transmission of these enteropathogenic species.

Disease has been identified as a major problem in the aquaculture industry for the welfare of the fish stocked as well as for its economic impact. The number of diseases affecting cultured fish has increased significantly during recent years with the emergence of several conditions that have added to the overall impact of disease on the industry. Frequently, a lack of scientific knowledge about these diseases is compounded by an absence of effective treatment and control strategies. This has been the case with rainbow trout gastroenteritis (RTGE), an emerging disease of rainbow trout (Oncorhynchus mykiss Walbaum). This study investigated several aspects related to its aetiology and control. A retrospective survey of UK rainbow trout farmers was undertaken to ascertain the extent and severity of RTGE in the UK as well as to identify RTGE risk factors at the site level. Participants in this study accounted for over 85% of UK rainbow trout production in 2004. It was found that the total number of RTGE-affected sites had risen from 2 in the year 2000 to 7 in 2005. The disease was only reported from sites producing more than 200 tonnes of trout/year for the table market. Analysis of risk factors associated with RTGE at the site level showed that this syndrome was associated with large tonnage and rapid production of rainbow trout for the table market. The data collected during this study enabled the identification of those sites that were most likely to present with RTGE the following year and this information was used to study the epidemiology of RTGE at the unit level. A prospective longitudinal study was undertaken in 12 RTGE-affected UK sites. It described in detail the impact, presentation, current control strategies and spread pattern of RTGE within affected UK sites. The risk factors associated with RTGE presence and severity were also investigated. Data were collected for each productive unit (i.e. cage, pond, raceway or tank) on the mortalities, fish origin, site management and environmental factors. RTGE was identified using a case definition based on gross pathological lesions. Analysis of these data revealed that RTGE behaved in an infectious manner. This conclusion was supported by the presence of a pattern typical of a propagating epidemic within affected units. Also, the risk of an unaffected unit becoming RTGE positive was increased if it had received fish from or was contiguous to a RTGE-affected unit. The presentation also suggested an incubation period of 20-25 days. Risk factor analysis identified management and environmental risk factors for RTGE, including high feed input and stressful events, which could be used to generate a list of control strategies. A study of the histopathological and ultrastructural presentation of RTGE was conducted. The location of segmented filamentous bacteria (SFB) and pathological changes found in affected fish were examined. Pyloric caeca were the digestive organ where SFB were found more frequently and in higher numbers, suggesting that this was the best location to detect SFB in RTGE-affected trout. Scanning and transmission electron microscopy revealed a previously undescribed interaction of SFB with the mucosa of distal intestine and pyloric caeca and this included the presence of attachment sites and SFB engulfment by enterocytes, as previously described in other host species. The SFB were not always adjacent to the pathological changes observed in the digestive tract of RTGE-affected trout. Such changes included cytoskeletal damage and osmotic imbalance of enterocytes, with frequent detachment. These observations suggested that if SFB are indeed the cause of RTGE their pathogenesis must involve the production of extracellular products. Analysis of the gross presentation and blood biochemistry in RTGE-affected fish was used to examine the patho-physiologic mechanisms of RTGE. To enable identification of positive RTGE cases for this study, a case definition was created from the information available on RTGE gross presentation in the literature. This case definition was assessed in a sample including 152 fish cases and 152 fish controls from 11 RTGE-affected UK sites, matched by unit of origin. The analysis of these fish using bacteriology, packed cell volume (PCV) and histopathology revealed that RTGE occurred simultaneously with other parasitic and bacterial diseases in a percentage of fish identified with this case definition. With the information gained after analysing the gross presentation, RTGE-affected fish without concurrent disease were selected for the study of the pathogenesis, which included blood biochemical analyses. These analyses revealed a severe osmotic imbalance, and a reduced albumin/globulin ratio suggesting selective loss of albumin, typical for a protein losing enteropathy. The role of the SFB “Candidatus arthromitus” in the aetiology of RTGE was assessed using a newly developed “C. arthromitus”-specific polymerase chain reaction assay (PCR) in conjunction with histological detection. This technique was applied to eight different groups of trout, including an RTGE-affected group and seven negative control groups. This analysis was conducted on DNA extracted from paraffin wax-embedded tissues as well as fresh intestinal contents. The results revealed the presence of “C. arthromitus” DNA in apparently healthy fish from sites where RTGE had never been reported. Additionally, SFB were observed histologically in two trout from an RTGE-free hatchery. These findings do not permit the exclusion of “C. arthromitus” as the aetiological agent for RTGE, although they suggest that the presence of these organisms in the digestive system of healthy trout is not sufficient to cause clinical disease, and therefore other factors are necessary. In conclusion, this study has used a multidisciplinary approach to the study of RTGE which has generated scientific information related to the epidemiology, pathogenesis and aetiology of this syndrome. The results of this project have suggested priority areas where further work is required, including experimental transmission of RTGE, field assessment of the control strategies proposed and further investigation into the aetiology of RTGE.

Thesis (PhD(Microbiology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Pathogenic Flavobacterium spp. cause serious disease outbreaks in a variety of farmed fish, which lead to large economic losses in the aquaculture industry on an annual basis. The ability of Flavobacterium johnsoniae-like isolates to grow as surface-associated communities (biofilms) in aquaculture systems poses a threat to fish health over extended periods of time. The biofilmforming ability of 28 F. johnsoniae-like isolates obtained from diseased fish were correlated with their chitin-degrading abilities and extracellular carbohydrate complexes (ECC) and their pulsed-field gel electrophoresis (PFGE) genotypes. Physiological changes in the proteome of 5 day planktonic, biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were analyzed by two-dimensional (2-D) gel electrophoresis and 17 differentially expressed and 14 uniquely expressed proteins were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Thirty-two differentially expressed genes in 5 day biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were identified using suppression subtractive hybridization (SSH). Significant negative correlations were observed between the chitin-degrading abilities and ECC and the biofilmforming capacity of 24 h biofilm cultures of F. johnsoniae-like isolates. Genetic heterogeneity was displayed by the F. johnsoniae-like isolates following PFGE. A significant positive correlation was observed between PFGE types and fish host species. Differentially and uniquely expressed proteins identified in planktonic, biofilm and agar surface-associated phases by 2-D/MS as well as differentially expressed genes identified in the biofilm and agar surface-associated phases by SSH were categorized as being involved in adaptation/protection, metabolic processes, membrane/transport/ motility and transcription/ translation. As far as we know, this is the first report on the characterization of differentially expressed genes and gene products of F. johnsoniae-like isolates obtained from diseased fish in South Africa.
AFRIKAANSE OPSOMMING: Patogene Flavobacterium spp. veroorsaak ernstige infeksie uitbrake in ’n verskeidenheid gekweekte vissoorte, wat jaarliks tot groot ekonomiese verliese in die akwakultuur bedryf lei. Die vermoë van Flavobacterium johnsoniae-tipe isolate om as oppervlak-gehegde gemeenskappe (biofilms) in akwakultuur sisteme te groei bedreig visgesondheid oor verlengde periodes. Die vermoë van 28 F. johnsoniae-tipe isolate om biofilms te vorm is vergelyk met hul vermoë om chitien te degradeer, die profiel van hul ekstrasellulêre koolhidraat komplekse (EKK) en bandpatrone verkry met puls-veld jel elektroforese (PVJE). Fisiologiese veranderinge in die proteoom van 5-dagoue planktoniese-, biofilm- en agar oppervlak-geassosieerde kulture van F. johnsoniae-tipe isolate YO12 en YO64 is met twee-dimensionele (2-D) jel elektroforese geanaliseer. Sewentien differensieël uitgedrukte en 14 uniek uitgedrukte proteïene is deur middel van matriks-geassisteerde laser desorpsie ioniserings-tyd van vlug-massa spektrometrie (MGLDI-TVV MS) geïdentifiseer. Twee-en-dertig differensieël uitgedrukte gene in 5-dag-oue biofilm- en agar oppervlak-geassosieerde kulture van F. johnsoniae-tipe isolate YO12 en YO64 was deur middel van suppressie afgetrokke hibridisasie (SAH) geïdentifiseer. Beduidende negatiewe korrelasies is tussen die chitin-degraderings vermoë en EKK en die biofilm-vormings kapasiteit van 24-uur-oue biofilm kulture van F. johnsoniae-tipe isolate waargeneem. Resultate verkry met PVJE het die heterogene samestelling van F. johnsoniae-tipe isolate uitgewys. ‘n Beduidende positiewe korrelasie is tussen PVJE groeperings en vis gasheer spesie waargeneem. Differensieël en uniek uitgedrukte gene geidentifiseer in die planktoniese-, biofilm- en agar oppervlak-geassosieerde fases is deur middel van 2-D/MS asook differensieël uitgedrukte gene geïdentifiseer in die biofilm en agar oppervlakgeassosieerde fases deur middel van SAH was as betrokke by aanpassing/beskerming, metaboliese prosesse, membraan/vervoer/ beweeglikheid en transkripsie/translasie gekategoriseer. Sover bekend is hierdie die eerste beskrywing van differensieël uitgedrukte gene en geenprodukte van F. johnsoniae-tipe isolate afkomstig van geinfekteerde vis in Suid Afrika.

[ES] La producción en acuicultura se ha visto menguada por aparición de enfermedades en los sistemas de cría de peces. En concreto, en la dorada (Sparus aurata), hay dos parásitos destacados: Enteromyxum leei (Myxozoa) y Enterospora nucleophila (Microsporidia). Hasta la fecha, para ninguno de los dos se ha establecido un cultivo in vitro, y solo para E. leei se ha conseguido establecer un modelo de mantenimiento de la infección in vivo. La presente tesis pretende incrementar el conocimiento sobre estos parásitos y sus relaciones con el hospedador, sentando las bases para generar soluciones que puedan ser aplicadas en la acuicultura. El objetivo con E. leei fue estudiar la inmunidad adquirida inducida en la dorada y la posibilidad de generar herramientas de diagnóstico y vacunas frente a esta enfermedad. Para ello, primero se demostró la resistencia del pez al parásito tras una segunda exposición, la cual duró hasta 16 meses. Además, la resistencia parece estar correlacionada con altos niveles de inmunoglobulina (Ig) M específica en sangre, y una alta expresión de Igs, incluso antes de la re-exposición al parásito. El siguiente paso fue afinar el protocolo de infección con E. leei. Los resultados mostraron que una semana es suficiente para transmitir la infección de E. leei por efluente, independientemente de la temperatura. Tras la demostración de la respuesta adaptativa eficaz frente a E. leei, y al disponer de un modelo de infección refinado, se realizó un ensayo de inmunización pasiva. Aquí, los resultados mostraron que los anticuerpos especi'ficos efectivamente consigue ralentizar la invasión del intestino por el parásito y disminuir los síntomas de la enfermedad. Paralelamente, el resultado del análisis del repertorio de las regiones variables de la IgM e IgT del intestino peces resistentes mostró la inducción de una respuesta policlonal en las ce'lulas B. En base a estos resultados, se realizó una búsqueda de antígenos de E. leei que pudieran ser utilizados como candidatos para la producción de vacunas (análisis proteómico) o herramientas de diagnóstico (análisis in silico). Para ello, se ensambló un transcriptoma de novo utilizando una muestra mixta de intestino de dorada y parásito. Los resultados dieron lugar a 7 y 12 candidatos en la búsqueda in silico y proteómica, respectivamente. En los estudios de E. nucleophila, debido a que fue descrita muy recientemente, el punto de partida fue más básico. Las muestras de este parásito solo se pueden obtener de brotes naturales en piscifactorias. Por ello, primero se realizó un estudio de caracterización de la patología de la infección a partir de peces infectados naturalmente. En etapas tempranas de la infección, el parásito se localiza principalmente en el intestino, pero meses después, la prevalencia en intestino baja e incrementa en los órganos hematopoyéticos y el esto'mago. Los signos clínicos de la infección consistieron en una reducción significativa del crecimiento, emaciación, y palidez de las paredes intestinales. A nivel celular, en los casos ma's graves se observó hipercelularidad en el epitelio intestinal y proliferación de ce'lulas rodlet, un elevado número de linfocitos en la base del epitelio e infiltración de granulocitos acidófilos en el epitelio intestinal. Finalmente se probaron varias formas de transmisión horizontal de E. nucleophila (cohabitación, efluente, intubación oral y anal) con para desarrollar un modelo de mantenimiento in vivo. Se consiguió la transmisión el parásito por todas las vías, pero con una disminución de prevalencia a lo largo del tiempo. Variables como la temperatura, la dosis, y el estado de los peces donantes parecen ser más determinantes que la ruta seleccionada para la transmisión. Entre las rutas probadas, la intubación anal parece ser la más prometedora, pero ninguna de ellas fue capaz de reproducir los signos clínicos observados en las infecciones naturales.
[CA] La producció en aqüicultura s'ha vist minvada per aparició de malalties en els sistemes de cria de peixos. En concret, en l'orada (Sparus aurata), hi ha dos paràsits destacats: Enteromyxum leei (Myxozoa) i Enterospora nucleophila (Microsporidia). Fins avui, per a cap dels dos s'ha establert un cultiu in vitro, i només per a E. leei s'ha aconseguit establir un model de manteniment de la infecció in vivo. La present tesi pretén incrementar el coneixement sobre aquests paràsits i les seves relacions amb l'hoste, establint les bases per a generar solucions que puguin ser aplicades en l'aqüicultura. L'objectiu amb E. leei va ser estudiar la immunitat adquirida induïda en l'orada i la possibilitat de generar eines de diagnòstic i vacunes enfront d'aquesta malaltia. Per a això, primer es va demostrar la resistència del peix al paràsit després d'una segona exposició, la qual va durar fins a 16 mesos. A més, la resistència sembla estar correlacionada amb alts nivells d'immunoglobulina (Ig) M específica en sang, i una alta expressió de Igs, fins i tot abans de la re-exposició al paràsit. El següent pas va ser afinar el protocol d'infecció amb E. leei. Els resultats van mostrar que una setmana és suficient per a transmetre la infecció de E. leei per efluent, independentment de la temperatura. Després de la demostració de la resposta adaptativa eficaç enfront de E. leei, i en disposar d'un model d'infecció refinat, es va realitzar un assaig d'immunització passiva. Aquí, els resultats van mostrar que els anticossos específics efectivament aconsegueix alentir la invasió de l'intestí pel paràsit i disminuir els símptomes de la malaltia. Paral·lelament, el resultat de l'anàlisi del repertori de les regions variables de la IgM i IgT de l'intestí peixos resistents va mostrar la inducció d'una resposta policlonal en les cèl·lules B. Sobre la base d'aquests resultats, es va realitzar una cerca d'antígens de E. leei que poguessin ser utilitzats com a candidats per a la producció de vacunes (anàlisis proteómico) o eines de diagnòstic (anàlisi in silico). Per a això, es va assemblar un transcriptoma de novo utilitzant una mostra mixta d'intestí d'orada i paràsit. Els resultats van donar lloc a 7 i 12 candidats en la cerca in silico i proteòmica, respectivament. En els estudis de E. nucleophila, pel fet que va ser descrita molt recentment, el punt de partida va ser més bàsic. Les mostres d'aquest paràsit només es poden obtenir de brots naturals en piscifactorias. Per això, primer es va realitzar un estudi de caracterització de la patologia de la infecció a partir de peixos infectats naturalment. En etapes primerenques de la infecció, el paràsit es localitza principalment en l'intestí, però mesos després, la prevalença en intestí baixa i incrementa en els òrgans hematopoètics i l'estómac. Els signes clínics de la infecció van consistir en una reducció significativa del creixement, emaciació, i pal·lidesa de les parets intestinals. A nivell cel·lular, en els casos més greus es va observar hipercelularidad en l'epiteli intestinal i proliferació de cèl·lules rodlet, un elevat nombre de limfòcits en la base de l'epiteli i infiltració de granulòcits acidòfils en l'epiteli intestinal. Finalment es van provar diverses formes de transmissió horitzontal de E. nucleophila (cohabitació, efluent, intubació oral i anal) amb per a desenvolupar un model de manteniment in vivo. Es va aconseguir la transmissió el paràsit per totes les vies, però amb una disminució de prevalença al llarg del temps. Variables com la temperatura, la dosi, i l'estat dels peixos donants semblen ser més determinants que la ruta seleccionada per a la transmissió. Entre les rutes provades, la intubació anal sembla ser la més prometedora, però cap d'elles va ser capaç de reproduir els signes clínics observats en les infeccions naturals.
[EN] Aquaculture production is hampered by the emergence of parasite diseases in fish farming systems. Among them, in Sparus aurata, there are two important enteric parasites described: Enteromyxum leei (Myxozoa) Enterospora nucleophila (Microsporidia). To date, no in vitro culture has been established for either parasite, and only for E. leei was it possible to establish a model for maintaining the infection in vivo. The aim of this thesis is to gain new knowledge about these parasites and their relationship with the host, also the basic foundations for generating solutions that can be applied in aquaculture. The general objective for E. leei was to study the acquired immunity induced in gilthead bream and the possibility of generating diagnostic tools and vaccines against this disease. To this end, resistance against the parasite was assessed with a second exposure against the parasite, which showed a resistance for at least 16 months. Besides resistance seemed to be correlated with high levels of specific immunoglobulin (Ig) M in blood, and a high expression of Igs, in particular, the soluble forms, even before re-exposure to the parasite. The next step was refining the protocol for effluent infection with E. leei by studying infection at different exposure time points, temperatures and population densities. The results showed that one week of exposure is sufficient to spread E. leei infection by effluent, regardless of temperature. After demonstrating the resistance against E. leei, and with a refined infection model, a passive immunization assay was performed. The results showed that the serum with specific antibodies effectively slows down the invasion of the gut by the parasite and reduces the symptoms of the disease. At the same time, the analysis of the repertoire of the variable regions of intestinal IgM and IgT showed an induction of a polyclonal response in B cells. On the basis of these results, a research was carried out for E. leei antigens that could have use as candidates for the production of vaccines (proteomic study) or diagnostic tools (in silico study) using the parasite transcriptomic data. To do this, a de novo transcriptome was assembled using a mixed sample of gilthead sea bream and parasite, with a posterior filtrate of the sequences. The In silico and proteomic analysis search resulted in 7 and 12 transcripts, respectively, which are being used for diagnostic and vaccine production. The starting point was more basic in E. nucleophila studies, since this is a recently described disease. The samples of this parasite can only be obtained from natural outbreaks in fish farms. Therefore, first study was carried out to characterize the pathology of the infection of naturally infected fish. In the early stages of the infection, the parasite is mainly located in the intestine, but months later, the prevalence is lower in the intestine and increases in the hematopoietic organs and the stomach. Clinical signs of infection were significant reduction in growth, wasting, and intestinal walls paleness. At the cellular level, in the most severe cases hypercellularity in the intestinal epithelium, proliferation of rodlet cells, high number of lymphocytes at the base of the epithelium and infiltration of acidophilic granulocytes in the intestinal epithelium were observed. Finally, horizontal transmission of E. nucleophila was tried using different transmission methods: cohabitation, effluent, and oral and anal intubation. Transmission of the parasite was achieved with all routes, but there was a decrease in prevalence over time in all cases except for the anal route. Variables such as temperature, dose, and the status of the donor fish appear to be more important than the selected route. Among the routes tested, anal intubation seemed to be the most promising, as it was sustained over a longer period of time, but none of them was able to reproduce the same clinical signs of infection observed in natural infections.
The authors kindly acknowledge the collaboration of anonymous fish farming companies allowing access to the animals during the disease outbreaks. We thank J. Monfort and L. Rodríguez (IATS-CSIC) for the technical assistance on histological processing.This work has been carried out with financial support from the European Union and the Spanish Ministry of Economy and Competitiveness (MINECO) under grant projects ParaFishControl (H2020-634429) and AGL2013-R-48560-C2-2-R, respectively. APS was contracted under ParaFishControl project. Primer sequences and access to the gilthead sea bream transcriptomic database were kindly provided by Prof. J. Pérez-Sánchez of the IATS- Nutrigenomics group. The authors thank I. Vicente for fish maintenance and technical assistance during samplings. The authors thank P. Boudinot (INRAE) for his help in designing and interpreting the immunoglobulin repertoire study and results, J. Pérez-Sánchez (IATS-CSIC) for providing access to the gilthead sea bream genome sequences to perform the repertoire analysis.This work was funded by the European Research Council (ERC Consolidator Grant 2016 725061 TEMUBLYM).
Picard Sánchez, MA. (2021). Control of enteric parasitic diseases of farmed gilthead sea bream: New insights into Enteromyxum leei (Myxozoa) and Enterospora nucleophila (Microsporidia) infections [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/167035
TESIS

Smith-Magenis Syndrome (SMS; OMIM #182290) is a multiple congenital abnormality and intellectual disability (ID) disorder caused by either an interstitial deletion of the 17p11.2 region containing the retinoic acid induced-1 (RAI1) gene or a mutation of the RAI1 gene. Individuals diagnosed with SMS typically present characteristics such as ID, self-injurious behavior, sleep disturbance, ocular and otolaryngological abnormalities, craniofacial and skeletal abnormalities, neurological and behavioral abnormalities, as well as other systemic defects and manifestations. Previous work by Vyas in 2009 showed temporal expression of rai1 in zebrafish embryos as early as 9 hpf. We hypothesize that there is maternal rai1 expression as early as zero hours post fertilization in wild type embryos. Using end-point PCR, we found that in fact there is maternal rai1 expression is detectable as early as 2 hours post fertilization (hpf) in wild type zebrafish embryos. Furthermore, we quantified rai1 expression using qPCR and found that rai1 expression declines significantly after 6 hpf. We hypothesize that a down regulation of rai1 or loss of rai1 will lead to morphological phenotypes, especially if that loss of rai1 function occurs during the earliest stages of zebrafish embryogenesis. Using a rai1morpholino oligonucleotide (MO), we found a loss of rai1 expression did not induce a morphological phenotype in in wild type embryos; furthermore, we also found that a loss of maternal rai1 expression did not induce a morphological phenotype as well. Utilizing a mutant rai1 zebrafish line, we found that both rai1 +/fh370 progeny nor rai1 fh370/fh370 progeny exhibited a morphological phenotype and that downstream targets such as bdnf were not affected by a reduction or complete loss of rai1. Prior research has shown that retinoic acid (RA) can induce rai1 expression. We hypothesize that RA can induce expression of rai1 during zebrafish embryogenesis. Using wild type fish and a rai1 in situ hybridization probe, we found that RA treatment at 25 hpf induced expression of rai1. The construction of a rai1 overexpression vector used for overexpression studies was started. Further development of GFP expression vector and zebrafish rai1 antibody are needed to determine if the morpholino is reducing rai1 protein expression.

This thesis describes a number of experiments designed to further our knowledge of the complex relationship that exists between nutrition and the immune system of fish, with a view to finding a practical means of improving disease resistance in farmed fish. In particular, vitamins are known to play an important role in vertebrate immune responses, and in the present study the practical value of vitamin C as an immunostimulant was investigated. A study was also made of the capacity of vitamin C to ameliorate stress-induced immunosuppression, and of the effect of stress on the demand for and distribution of vitamin C amongst various tissues of Atlantic salmon (Salmo salar L). Taken together, the results of these studies revealed no significant evidence for a fundamental role for vitamin C in regulating the stress response of salmon, and no significant evidence that high vitamin C diets have any value as immunostimulants in aquaculture. The practical value of diets containing high levels of vitamin A and carotenoids were also investigated. Whilst there was some evidence that vitamin A deficiency resulted in increased disease susceptibility, there was no evidence to suggest that feeding elevated levels of these compounds was immunostimulatory. Hence, the practical value of these compounds in promoting disease resistance in aquaculture also appears limited. The immunomodulatory effect of differential food acquisition by individual fish kept in different ration groups (i.e. as determined by management feeding strategy), and also within those ration groups (as determined by social interaction) was also investigated. The results obtained indicate that feeding very high ration levels may have deleterious consequences for disease resistance in salmonids, a finding which may have major implications for the aquaculture industry. Overall, the results presented in this thesis have served to eliminate several avenues of research which were popular at the time of its conception, and to suggest new approaches which may lead to real improvements in disease resistance of farmed fish in the future.

Orientador: Silvia Regina Brandalise
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Introdução Os osteossarcomas (OS) são tumores agressivos, primários de osso, com prognóstico reservado. As deleções dos genes supressores de tumor, RBl e P53, localizados nos cromossomos 13 e 17 respectivamente, são freqüentemente encontradas neste tipo de tumor. As alterações citogenéticas encontradas nos OS são de alta complexidade, porém nenhuma delas é recorrente, não podendo caracterizá-lo. A técnica da Hibridização Genômica Comparativa (CGH) é uma ferramenta muito precisa para o estudo das deleções e amplificações gênicas ocorridas neste tumor. Materiais e Métodos. Tecido tumoral de 41 crianças com OS foi analisado pela técnica de CGH para pesquisar possíveis ganhos e/ou perdas gênicas . A técnica da Hibridização In Situ Fluorescente (FISH) foi realizada para estudar as deleções dos genes P53 e RBl. Vinte e quatro pacientes eram do sexo feminino e 17 do sexo masculino, com mediana de 12 anos e 4 meses. Resultados. As anormalidades cromossômicas observadas com a técnica de CGH foram diversas e variadas, especialmente ganhos nos cromossomos lp, 2p, 3q, 5q,5p e 6p e, perdas nos cromossomos 14q (50% no - 14q 11.2), 15q e 16p. Alto índice de perdas foi observado no cromossomo 21 (26 de 41 casos; p=0,008), sendo a região mais freqüentemente afetada a 21ql 1.2. Com relação ao estudo dos supressores tumorais, a deleção do P53 ocorreu em 68,3% dos casos (p=0,02) e do RBl em 87,5% dos casos (p=0,000001). Conclusão. Apesar de ambos supressores (PS3 e RBl) estarem deletados na maioria dos pacientes, este evento parece não estar associado ao prognóstico. Anormalidades ainda não reportadas presentes no cromossomo 21 nos OS pediátricos, sugerem que a seqüência mapeada nesta região cromossômica possa estar envolvida na patogênese deste tumor
Abstract: Background. Osteosarcomas (OS) are aggressive bone tumors and often have a poor prognosis. It is already known that abnormalities in chromosomes 13 and 17 are frequently observed in OS patients, being also expected a deletion of RBI and P53 genes. The tumors exhibit karyotypes with a high degree of complexity, that has made it difficult to determine if any recurrent chromosomal aberrations could characterize OS. To address inherent difficulties associated with classical cytogenetic analysis, comparative genomic hybridization (CGH) has been applied to OS tissue. Patients and Methods. Forty one pediatric OS specimens were analyzed by CGH techniques, and the expression of RBI and P53 were analyzed by FISH . Twenty four patients were girls and 17 boys. Median age was 12 years and 4 months.Results. Chromosomal abnormalities were highly diverse and variable specially gains in chromosome lp, 2p, 3q, 5q , 5p and 6p and losses in chromosome 14q (50% in - 14q 11.2), 15q and 16p. High level of losses in chromosome 21 were present (26 of 41 cases; p-0,008), being 21 q 11.2 region the most frequent one. Concerning about genes expression, P53 is deleted in 68,3% of the cases (p=0,02) and RBI in 87,5% (p=0,000001) .Conclusion. Although both oncogenes (P53 and RBI) are deleted in OS population, it remains impossible to determine if this abnormality is a prognostic factor. These new and unreported findings in chromosome 21 of pediatric OS tumors, suggest that specific sequences mapping these chromosomal regions, would likely to play a role in the development of OS
Doutorado
Pediatria
Mestre em Saude da Criança e do Adolescente