Dissertations / Theses on the topic 'Fish immunology'

Given the widespread concern that chemical contaminants may be associated with infectious disease outbreaks in marine fish populations, work has been undertaken with the aim of developing a suite of non-specific and specific assays of marine fish immune function and the application of these techniques in a variety of field and laboratory investigations. Most of the work focused on dab, Limanda limanda (L.) in view of the importance of this species in several North Sea fish disease monitoring programmes, and was also supplemented with investigations of specific immune function in turbot, Scopthalmus maximus (L.). Initial field studies examined non-specific immune function in terms of lymphoid organ morphology in dab sampled along a North Sea gradient of chemical contamination during the March 1990 ICES/IOC Bremerhaven workshop. Significant differences were observed in the kidney and spleen cell populations from dab, and these observations were considered in view of the various other physico-chemical and biological results generated during the Bremerhaven workshop. Following the valuable experience gained of the practical aspects of the field monitoring approach, laboratory investigations were initiated with the aim of developing a suite of immune function assays for deployment in either laboratory or field studies of marine fish health. Assays for non-specific immune functions were considered, including serum protein and lysozyme levels, methods of phagocyte collection, phagocyte chemiluminescence, calorimetric detection of individual reactive oxygen species and in vitro cell migration assays. Additional field work was undertaken, with the monitoring of serum total protein levels and lysozyme activity in dab sampled from Lyme Bay, UK This study provided evidence of a marked seasonal variation in non-specific immune function, which appeared to be associated with environmental factors (e.g. water temperature) and the reproductive cycle. Selected non-specific assays were applied to dab and turbot exposed under controlled laboratory conditions to a variety of important marine contaminants, including cadmium, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), and the biological data integrated with appropriate chemical characterisation of the exposure matrix. Using cultured turbot as a test species, the non-specific assays were also supplemented with the assay of specific immune function in fish exposed to PCB contaminated sediments. In brief, there was evidence of significant impairment of immune function in fish exposed to either individual contaminants (viz. cadmium and PAHs) or contaminant mixtures (viz. PAHs and PCBs) under the laboratory conditions described. In summary, the project was successful in its primary aims of developing a suite of techniques for evaluating both cellular and humoral immune functions in marine flatfish, and applying these techniques in the laboratory to assess the impact of important classes of environmental contaminants on fish health. Selected techniques were also used in field monitoring studies of marine fish immune function, illustrating the potential of such techniques for use in future laboratory and field studies of fish health.

Singular or dual dietary depletions of vitamins C and E in Atlantic salmon parr increased susceptibility to Aeromonas salmonicida challenge. An array of immune parameters were evaluated to identify the involvement of these vitamin depletions on the immune response. Dietary vitamin E levels in salmon had no impact on haematological parameters, total serum protein or lysozyme levels. Similarly, dietary vitamin E levels did not affect leucocyte antibody production, macrophage activating factor (MAF) release and respiratory burst (RB) phenomena. However, haemolytic and opsonic properties of complement were compromised in vitamin E depleted salmon. Parenteral administration of vitamin E to vitamin E depleted carp did not elevate phytohaemagglutinin (PHA) lymphocyte proliferation responses or complement activity. In vitro additions of vitamin E to lymphocytes from carp fed a commercial diet did not elevate PHA proliferation responses either. The increased disease susceptibility provoked by dietary vitamin C restriction in Atlantic salmon was not correlated with serum protein levels, differential leucocyte numbers or phagocyte functions as tested by RB activity or phagocytosis by macrophages. Lymphocyte functions were operational in these fish as examined by MAF secretion and antibody production. Analogous to vitamin E depletion, dietary vitamin C restriction in salmon compromised complement haemolytic activity. Elevating the vitamin C content of diets above normal levels enhanced complement activity in salmon. Vitamin C was a potent modulator of rainbow trout leucocyte functions. In vitro supplementation of vitamin C in the sodium (NaAsc) or polyphosphate (PPAsc) form was required for PHA-proliferation responses. MAF secretion was also augmented by in vitro additions of NaAsc to leucocytes obtained from vitamin C depleted trout. Injecting NaAsc into depleted fish elevated PHA proliferation responses compared with saline-injected controls. Leucocytes from the latter group could recover proliferative responses to levels associated with NaAsc-injected fish with in vitro additions of 1x10-3 and 1x10-4M PPAsc. Increased disease susceptibility, reduced complement activity and haematocrit values were symptomatic of combined dietary vitamin C and E depletion in salmon parr. Total and differential leucocyte numbers, or serum parameters including antiprotease activity and total protein level were unaffected by this dietary regime. Although poorly adherent, macrophages obtained from dually depleted salmon had similar RB values and expressed greater responsiveness to a MAF-containing supernatant than vitamin sufficient counterparts. Lymphocyte functions were impervious to dual vitamin depletion as antibody and MAF production responses were intact. In vitro additions of NaAsc and PPAsc were shown to elevate RB responses in macrophages from vitamin-restricted and -adequate salmon. MAF secretion was demonstrated to be a temperature-dependent phenomenon in rainbow trout leucocytes. After 48h acclimation at low in vitro temperatures (6 C), leucocytes obtained from trout at 14C expressed impoverished MAF production. Acclimation of trout to 7 C did riot rescue MAF production. Also, normal RB activity was reduced in macrophages obtained from fish at 14 C after 48h at 6 C in vitro. However, if allowed to acclimate, these macrophages become more responsive to MAF and recovered RB activity to levels associated with macrophages held at 10 and 18C. RB responses of macrophages from fish at 7 C functioned equally well across a wide range of in vitro temperature regimes. Phagocytic activity of macrophages obtained from fish at 14 C and placed at 6 C in vitro for 48h expressed temperature sensitivity. This was especially apparent in fish fed a dietary ?-3/?-6 fatty acid ratio of 2.0. However, this dietary regime also elevated phagocytic activity of these macrophages to such an extent, that their response surpassed that of macrophages from fish fed commercial diets or ?-3/?-6 ratios of 0.5 and 1 even at low in vitro temperatures.

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 Murray-Darling basin is the largest river system in Australia with significant economic, social, recreational and cultural value. It supplies water for drinking and agriculture to a large inland area of the eastern and southern states of Australia. It is also the ultimate sink for many environmental contaminants that result from human activities within the catchment. Aquatic organisms live intimately with their environment and may be continuously exposed to these contaminants through the water column or the food chain. Some chemicals are bioaccumulated and biomagnified in tissue to reach high body burdens. Populations of native fish species within the Murray-Darling basin have been in decline since human settlement, yet little is known about the lethal and sublethal effects of environmental pollutants on native freshwater fish and many of the Australian water quality guidelines are based on data from exotic fish species. Researchers have correlated levels of pollution with immune dysfunction and an increased incidence of disease amongst wildlife populations. Many of the pollutants of the Murray-Darling basin have known immunotoxicity in both mammals and exotic fish species. The immune system is a sensitive target organ because, in order to maintain integrity, it requires constant renewal through the rapid proliferation and differentiation of cells. Efforts to increase numbers of native fish in the wild have led to an aquaculture industry that produces fingerlings for the restocking of waterways. In more recent years, this industry has matured and now produces table-size native freshwater fish for local and international markets. Although the industry has researched areas of reproduction, nutrition and stocking, there is little understanding of the immunology or immunotoxicology of Australian freshwater fish. This research project investigated the immunology of three large native fish species (i.e. 2 Murray cod, golden perch and silver perch), which are the basis of the native freshwater aquaculture industry. Additionally, a small fish species native to the basin (i.e. crimsonspotted rainbowfish) was studied as an alternative to the use of large fish. Of the four species, Murray cod possessed characteristics that made it an excellent candidate for ecoimmunotoxicity testing.

Aquesta tesis doctoral està basada en estudiar la resposta immunològica dels peixos en models d'estrès i d'activació del sistema immune des la genòmica funcional. L'aplicació de tecnologies moleculars com el Differential Display van permetre identificar y clonar por primera vegada en orades (Sparus aurata) y en altres especies de peix, el gen enolasa. Aquest enzim glucolític s'ha plantejat per primera vegada com un bon marcador molecular per estudiar el benestar dels peixos. Per mitjà de l'ús d'una plataforma de microarrays dissenyada específicament per a salmònids, i altres metodologies biomoleculars, es va comprovar que els nivells d'enolasa eren regulats en diferents teixits y en diferents especies de peix, com també en adverses situacions per l'animal. D'altra banda, s'han estudiat diferents gens immunològics candidats a ser possibles gens per l'estudi del sistema immunològic dels peixos. Aquests gens s'han estudiat a nivell d'expressió en teixits de truites (Oncorhynchus mykiss) mitjançant PCR convencional i PCR quantitativa, i l'ús de metodologies biomoleculars i bioinformàtiques. Entre ells, destaca el factor de transcripció PU.1, un gen indispensable per el desenvolupament de l'hematopoesi. Aquest gen, s'ha clonat i caracteritzat per primera vegada en salmònids. L'expressió de PU.1 s'ha estudiat mitjançant l'ús d'hibridacions in situ en ronyó anterior y en cervell de truita. A més, l'ús de microarray en aquest dos teixits han permès fer un estudi exhaustiu i pioner a nivell de transcriptòmica en peixos. Les anàlisis del xip de microarray, ha revelat que grups de gens s'activen o s'inhibeixen com a conseqüència d'un estrès immunològic.
En resum, aquesta tesis doctoral ha aplicat el desenvolupament de noves tecnologies moleculars pioneres en peixos, com el microarray, la clonació de noves seqüències gèniques i la bioinformàtica, per estudiar la genòmica funcional dels peixos en situacions d'activació dels mecanismes d'estrès i del sistema immune.
The main results of the present thesis can be integrated to a better understanding the stress and the immune responses in fish at a transcriptional level. The application of functional genomic tools, which encloses from using simple PCR analysis to more modern, sophisticate and fashionable microarray technique, allowed us to identified transcriptional regulations of certain set of genes which are enhanced or repressed under stress conditions. Our findings contribute to increase knowledge of molecular mechanism involved in coping the stress and immune responses in fish and provides a better understanding of fish physiology when fish health is threatened. Furthermore, thesis results may be interesting for aquaculture which looks for good biomolecular markers that may improve fish production and fish quality. The isolation, characterization and gene expression study with further microarray analysis of the enolase gene, allowed us to describe enolase as a possible biomolecular marker to determine fish welfare. The in situ hybridization study of the hematopoietic transcription factor PU.1, contributed to amplify the knowledge of the development of the fish immune system. Throughout this thesis, DNA sequences and mRNA expression levels of several genes studied, have contributed to enlarged genomic fish database.
In summary, this thesis described from a transcriptional level, gene expression and molecular mechanisms activated or repressed when fish welfare is threatened and contributes to a better understanding of transcriptiomic mechanisms required to cope with the stress.

Peressigsäure (PES) hat seit kurzem als Desinfektionsmittel in der Aquakultur Einzug gehalten. Gegenüber anderen konventionellen Desinfektionsmitteln besitzt es in niedrigsten Konzentrationen (ca. 1 mg l-1) eine hohe Effektivität. Des Weiteren hat die Anwendung von PES kaum einen negativen Einfluss auf die Umwelt. Die Applikation von PES in Aquakulturanlagen erfolgt direkt über das umgebende Haltungswasser. Dies geht mit einem direkten Kontakt der Mikroorganismen und der Fische mit dem Wirkstoff einher. Aus diesem Grund ist generell ein Einfluss auf die Fischgesundheit und die Wasserqualität zu erwarten. Dieser hypothetische Einfluss ist bislang jedoch unzureichend untersucht worden. In der Praxis werden zumeist zwei Applikationsstrategien verfolgt: 1. Wiederholende Kurzzeitpulsapplikationen mit relative hohen PES-Konzentrationen (1-2 mg l-1) und 2. Die kontinuierliche Applikation mit relative geringen PES-Konzentrationen (≤ 0,2 mg l-1) in der Wasserzufuhr. Die potentiellen Unterschiede dieser zwei Strategien speziell auf die Fischgesundheit und die Wasserqualität sind bislang unklar. In der vorliegenden Studie wurden Effekte einer PES-Applikation auf die Fischgesundheit und die mikrobielle Aktivität in identischen Durchflussystemen untersucht. Diese Systeme garantieren eine optimale Wasserqualität. Regenbogenforellen wurde als Testorganismen gewählt. Verschiedene Stressparameter, Parameter des oxidativen Stresses, Wachstum, Kiemenhistologie und Parameter der angeborenen Immunantwort wurden zur Bewertung der Fischgesundheit herangezogen. Sauerstoff, pH und die visuelle Biofilmformation wurden kontinuierlich kontrolliert um die mikrobielle Aktivität zu interpretieren. Dazu wurde zweimal wöchentlich mit 1 mg l-1 PES (Pulsbehandlungen) im Haltungswasser und kontinuierlich mit 0,2 mg l-1 PES am Zulauf exponiert und verglichen. Die Ergebnisse belegen, dass die Pulsapplikationen mit 1 mg l-1 PES, im Gegensatz zur kontinuierlichen Applikation mit 0,2 mg l-1 PES die Fische stressten. Die Fische adaptierten sich jedoch an die PES-Pulsapplikationen. Dies wurde durch nachfolgend weniger heftige Reaktionen der Fische post applicationem, reduzierte Kortisolausschüttungen und unveränderte Reaktionen auf andere Stressoren deutlich. Obwohl die PES-Applikation leichte Hyperplasien in den Kiemen induzierte, war kein Einfluss auf das Wachstum und die angeborene Immunantwort feststellbar. Dies kann als ein Beleg für den fehlenden Einfluss der PES-Exposition auf die Fischgesundheit bewertet werden. PES induzierte unabhängig von den Applikationsstrategien oxidativen Stress in den Fischen. Als Antwort auf die PES-Applikation steigerten die Fische ihre antioxidative Antwort gegen die freien Sauerstoffradikale speziell in den Kiemen und im Serum. Unabhängig von den extrem geringen PES-Konzentrationen in der kontinuierlichen Applikation wiesen die Fische einen geringen oxidativen Stress auf. Der oxidative Stress der Fische in der Pulsexposition war hingegen periodisch nachweisbar. Daraus ist zu schlussfolgern, dass die Fische zwischen den Pulsexpositionen, wenn keine PES vorhanden war, Phasen der Erholung hatten. Das Fehlen dieser Erholungsphase in der kontinuierlichen Expositionsgruppe führte zu einer signifikanten Reduktion der Antiprotease-Aktivität im Serum. Dies impliziert das Risiko einer chronischen Entzündung. Die antimikrobiellen Effekte sind stark von der PES-Konzentration abhängig. Die Pulsapplikation mit 1 mg l-1 PES hemmte die mikrobielle Aktivität stärker als die kontinuierliche Exposition durch einen stärkeren oxidativen Stress. Dadurch wurde der Biofilm fast vollständig erodiert, und die mikrobielle Sauerstoffsverbrauch und nitrifikation inhibiert. Die PES-Konzentrationen in der kontinuierlichen Exposition waren zu gering um signifikante Effekte auf den Mikroorganismen auszuüben. Des Weiteren kann das PES-Zerfallsprodukte, die Essigsäure und Acetate, eine potentielle Kohlenstoffquelle für die Mikrobiota darstellen. Der daraus resultierende stärkere Biofilm kann durch die Besiedelung mit fakultativen Fischpathogenen eine Gefahr für die Fischgesundheit darstellen. Auf Grund des starken antimikrobiellen Effekts und des geringen Risikos die Fischgesundheit zu beeinträchtigen, werden periodisch regelmäßige PES-Applikationen in Konzentrationen von 1-2 mg l-1 empfohlen. Effekte einer PES-Applikation auf Spiegelkarpfen und die Wasserqualität in stark belastetem Wasser einer geschlossenen Aquakulturkreislaufanlage (RAS) wurde ebenso untersucht. Die Induktion einer schlechten Wasserqualität erfolge durch den Stopp der Wasserzufuhr zu den Tanks. Simultan zu den Wasserstopps erfolgte eine Applikation mit 1 mg l -1 PES. Die Stressantwort, Kiemenhistologie und die angeborene Immunantwort wurde mit nicht mit PES exponierten Kontrollfischen verglichen. Der Stopp der Wasserzufuhr steigerte die gesamte heterotrophen Bakteriendichte (GHBD) auf das Sechsfache. Im Gegensatz dazu wurde in den Expositionsgruppen die GHBD um 90% gesenkt. Der stark mikrobiozide Effekt der PES-Exposition verbesserte die Gesundheit der Kiemen, verhinderte bakterielle Infektionen welche in den Kontrollgruppen kurzzeitig festgestellt wurden. Zusammenfassend erhält PES appliziert periodisch in Konzentrationen von 1-2 mg l-1, im Fall der optimalen Wasserqualität, die gute Wasserqualität mit geringfügiger Beeinträchtigung der Fischgesundheit. In der Aquakulturproduktion, in welcher die Wasserqualität meistens durch die hoher Besatzdichte und organischer Belastung verschlechtert wird, verhindern regelmäßige prophylaktische PES-Applikationen Infektionen und begünstigen die Fischgesundheit.
Peracetic acid (PAA) has been recently introduced to aquaculture as a sustainable disinfectant. It has great advantages over conventional disinfectants by having high effectiveness and low environmental impact at very low concentrations (around 1 mg L-1). The application of PAA in aquaculture facilities is realized by adding PAA products to the rearing water. This leads to unavoidable exposure of fish and microorganisms (surface-attached and waterborne) to PAA. Consequently, a potential impact of PAA on fish health and microbial activities is expected. This potential impact, however, has been poorly studied. In aquaculture practice, two strategies are broadly used to apply PAA: short term high dose (1-2 mg L-1 PAA) periodic/pulse applications or continuous low dose (≤ 0.2 mg L-1 PAA) application related to the makeup water flow. The potential difference between these two strategies remains unclear, especially concerning their impacts on fish health and water quality. In the present study, the impact of PAA on fish health and microbial activities was tested in identical flow-through systems controlled with optimal water quality. Rainbow trout was selected as the model fish. Various parameters of stress, oxidative stress, growth, gill histology and innate cellular/humoral immunity were measured to indicate fish health. Oxygen, pH and visible biofilm formation were continuously monitored to interpret changes of microbial activities. In addition, the application strategies, biweekly pulse applications of 1 mg L-1 PAA in the rearing water and the continuous application of 0.2 mg L-1 PAA in the inflow, were compared. The results indicate that pulse applications of 1 mg L-1 PAA stressed the naïve fish during the first exposure, while the continuous application not. Fish could progressively adapt to PAA-induced stress, as indicated by less intensive behavioral reaction, reduced cortisol release and unaffected response to another stressor. Although the exposure to PAA induced slight hyperplasia in fish gill, the growth and innate immunity were affected, indicating unaffected overall health. PAA induced oxidative stress in fish, regardless of the application strategies. In response, fish enhanced their antioxidative defense, especially in gill and serum, to scavenge excessive free radicals induced by exposure to PAA. Despite of extremely low PAA concentration measured during the continuous application, the constant input of PAA induced a constant mild oxidative stress to fish. In contrast, the oxidative stress induced by pulse 1 mg L-1 PAA applications was periodic present. Consequently, fish had periodic recovery phases when the pulse PAA applications were absent. The lack of recovery phases in fish exposed to the continuous PAA application resulted in a significant reduction of antiprotease activity in serum. This implies a potential risk of chronic inflammation. The antimicrobial effect of PAA depended on applied concentration. The pulse applications of 1 mg L-1 PAA strongly inhibit microbial activities by inducing a strong oxidative stress. As a result, the biofilm in fish tanks was nearly erased, and the microbial oxygen consumption and nitrification were inhibited. In contrast, the PAA concentration during the continuous application was so low that only a minor antimicrobial effect was observed. In addition, the degradation products, acetic acid and acetate, were beneficial for the biofilm formation by providing organic carbon. The enhanced biofilm may become a potential risk by providing protective shed for opportunistic pathogens. Due to the strong antimicrobial effect and low risk to affect fish health, it’s recommended to apply PAA periodic at high concentrations (1-2 mg L-1) with sufficient intervals. The impact of PAA on fish health and water quality was also tested in a mirror carp recirculating aquaculture system (RAS) challenged with bad water quality. The challenge of bad water quality was realized by transient water stops in fish tanks. Simultaneous to the transient water stops, PAA at 1 mg L-1 was applied. The stress, gill histology and innate cellular immunity were compared in fish with or without simultaneous PAA treatments. The transient water stops caused a 6-fold increase of heterotrophic bacterial density in water, while the simultaneous PAA treatments caused a 90% decrease of heterotrophic bacterial density. The strong antibacterial effect of PAA significantly improved the gill health of fish, and effectively prevented bacterial infections, which were short-term present in fish exposed to transient water stops alone. To sum up, PAA applied periodically at 1-2 mg L-1 in optimal water quality is effective to maintain the water quality at a low cost of scarifying fish health. In production-scale aquaculture facilities, where the water quality is often deteriorated by high stocking density and organic load, regular applications of PAA are especially beneficial to enhance fish health and prevent potential infections.

Commercial capture-based aquaculture of the Atlantic bluefin tuna (ABT), Thunnus thynnus (L.), has been prominent in the Mediterranean for over a decade. Owing to several limitations encountered in working with the species, including its high commercial value, there has been little research carried out relating to this species. The objective of this study was to examine several health parameters of captive ABT. These included an examination of coronary artery lesions, haematology, plasma biochemistry, assessment of immune function and changes in fatty acid (FA) flesh content through the on-growing period. Arteriosclerosis in fish is a pathologic condition of uncertain etiology and involves the main coronary artery in teleosts. Apart from reports of their widespread occurrence in salmonids, they have been described from a restricted number of wild ABT specimens but have not received further attention. This investigation analysed the effect of size and period of net-pen rearing on the prevalence and severity of arteriosclerotic lesions in ABT. Coronary arteries from wild and captive fish were investigated and prevalence was 100 %, but increasing structural degradation was observed with increasing fish size, suggesting that lesions progress throughout the life of the fish. Due to the limited availability of wild specimens, the effect of captivity on arteriosclerosis in ABT could not be adequately quantified, although observations suggest that the farming process has no major effect on arteriosclerotic lesions in ABT. Studies on the haematology, plasma biochemistry and immunology of ABT are limited. Haematological and plasma biochemical indices are useful in animal health assessment but use of these requires the establishment of species-specific ranges. Blood was collected from captive ABT specimens of varying weight (61-361 kg) and the major haematological (n = 45), plasma biochemical (n = 30) and immunological parameters (n = 45) were quantified. Size-based differences were found in haematological indices between experimental sub-groups including increased erythrocyte number and haemoglobin level in smaller ABT. No differences were found in immunological parameters except for total IgM levels, which were higher in the smaller individuals. Preliminary investigations indicated that disease prevalence in captive ABT is very low. Epidermal mucus is an important interface between fish and their environment and comprises immunological components which act as a first barrier against pathogen entry or colonisation. Mucus was collected from captive ABT and analysed for innate immune components. The presence of IgM was detected in the mucus of ABT by an enzyme-linked immunosorbent assay and several different enzymes were detected with an API-ZYM kit assay. Zymography experiments confirmed the presence of protease-like enzymes in the mucus, while enzyme assays quantified alkaline phosphatase, protease, esterase and cathepsin B activities. Lysozyme levels were high. The mucus agglutinated sheep erythrocytes but did not demonstrate complement or bacteriolytic activity. There is restricted information on the fatty acid composition of farmed ABT or how this is influenced when the fish are held under commercial aquaculture conditions. This study investigated the FA composition of farmed ABT, its variation by dorsal muscle region and the correlation between dietary FA composition with that of the fish. Analysis of flesh samples retrieved from farmed ABT did not reveal significant differences in the FA composition of experimental sub-groups irrespective of size, time held in captivity or diet. These results indicate that FA metabolism in ABT is substrate-selective. Gene expression measurements from several organs of ABT showed that expression of Δfad5 and elovl5, genes involved in FA metabolism, were highest in the brain followed by the liver but no expression of these genes was detected in the spleen. The findings of this research address aspects of health evaluation and nutritional status in farmed ABT and are discussed in terms of farming practice. Conclusions from some of these studies suggest that the practice of holding wild-captured stock in cages for periods of up to 18 months does not result in significant impact on ABT.

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O aparecimento de doenças em peixes de criação intensiva é um problema enfrentado no Brasil e no mundo. Algumas substâncias podem influenciar as respostas do sistema imune de peixes, como o levamisol, através da modulação de parâmetros imunes, aumentando a resistência contra diversos agentes. Foram avaliados os efeitos do levamisol na dieta em dois experimentos. O primeiro experimento avaliou a suplementação com 0, 125, 250 e 500 mg kg-1 do imunoestimulante administrado por sete e 15 dias, no qual foram avaliados os parâmetros da imunidade inata, hematológicos e bioquímicos. A administração do levamisol por sete e 15 dias promoveu alterações em parâmetros imunológicos e hematológicos. A administração do levamisol pode promover imunomodulação, entretanto a determinação do efeito não ficou claro devido às respostas contraditórias em cada parâmetro avaliado. O segundo experimento consistiu em suplementação com 0, 125, 250 e 500 mg kg-1 do imunoestimulante administrada por sete dias conjuntamente com a imunização com bactérias Aeromonas hydrophila. A imunização e a administração de levamisol promoveram aumento do título de anticorpos, atividade bactericida do soro, hematócrito, número de eritrócitos, leucócitos totais e trombócitos nos pacus. A administração de levamisol por sete dias e a imunização de pacus promoveu melhora de alguns parâmetros da imunidade adquirida e inata de defesa. Entretanto outros protocolos devem ser estudados para avaliar o efeito do levamisol sobre o sistema imune de pacu
The emergence of diseases in fish farming is a problem faced in Brazil and worldwide. Some substances can influence the immune system responses of fish, like levamisole, increasing resistance against various etiological agents. Were evaluated the effects of levamisole in diet in two experiments. The first experiment evaluated supplementation with 0, 125, 250 and 500 mg kg-1 imunoestimulante administered by seven and 15 days in which have been assessed the parameters of innate immunity, haematological and biochemists. The administration of levamisole by seven and 15 days promoted changes in haematological and immunological parameters. The administration of levamisole can promote immunodulation, however the determination of the effect not clear due to contradictory answers evaluated in each parameter. The second experiment consisted of supplementation with 0, 125, 250 and 500 mg kg-1 imunoestimulante administered by seven days together with immunization with inactivated Aeromonas hydrophila bacteria with formaldehyde. Immunization and administration of levamisole promoted increase antibody titre, serum bactericidal activity, hematocrit, number of erythrocytes, leucocytes and thrombocytes in pacus totals. Administration of levamisole for seven days and immunization pacus promoted improves some parameters of innate and acquired immunity defense. However other protocols must be studied to assess the effect of levamisole on the immune system of pacu

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.

Mycobacterium marinum is an established surrogate pathogen for Mycobacterium tuberculosis because of M. marinum 's strong conservation of thousands of orthologous genes, lower risk, lower financial burden to researchers, and similar pathology in fish. This pathogen causes TB-like chronic disease in a wide variety of fish species and can mount superficial infection of human tissues. As in human TB, the microbe grows within the host macrophages, can mount life-long chronic infections, and produces granulomatous lesions in target organs. One of the fish species known to manifest chronic "fish TB" is the small laboratory fish, Japanese medaka (Oryzias latipes). Recently, our lab documented the progression of the bacterium from the lumen of the gut to underlying tissues and to target organs to mount infection. Since the bacterium can be observed crossing the epithelia to mount infection, I tested to see if mucosal immunity against a wild-type challenge could be induced by initially priming the fish to a live, attenuated vaccine strain. This thesis demonstrates that inoculation by ingestion is an efficient mode by which medaka can become infected and vaccinated with M. marinum. Furthermore, my thesis shows that orally vaccinating fish with a live, attenuated strain indeed provides protection in the gut, liver, and kidney against a virulent, wild-type challenge.

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).

Orientadora: Elisabeth Criscuolo Urbinati
Banca: José Eurico Possebon Cyrino
Banca: Helio José Montassier
Banca: Janessa Sampaio de Abreu
Banca: Fabiana Pilarski
Resumo: O aparecimento de doenças em peixes de criação intensiva é um problema enfrentado no Brasil e no mundo. Algumas substâncias podem influenciar as respostas do sistema imune de peixes, como o levamisol, através da modulação de parâmetros imunes, aumentando a resistência contra diversos agentes. Foram avaliados os efeitos do levamisol na dieta em dois experimentos. O primeiro experimento avaliou a suplementação com 0, 125, 250 e 500 mg kg-1 do imunoestimulante administrado por sete e 15 dias, no qual foram avaliados os parâmetros da imunidade inata, hematológicos e bioquímicos. A administração do levamisol por sete e 15 dias promoveu alterações em parâmetros imunológicos e hematológicos. A administração do levamisol pode promover imunomodulação, entretanto a determinação do efeito não ficou claro devido às respostas contraditórias em cada parâmetro avaliado. O segundo experimento consistiu em suplementação com 0, 125, 250 e 500 mg kg-1 do imunoestimulante administrada por sete dias conjuntamente com a imunização com bactérias Aeromonas hydrophila. A imunização e a administração de levamisol promoveram aumento do título de anticorpos, atividade bactericida do soro, hematócrito, número de eritrócitos, leucócitos totais e trombócitos nos pacus. A administração de levamisol por sete dias e a imunização de pacus promoveu melhora de alguns parâmetros da imunidade adquirida e inata de defesa. Entretanto outros protocolos devem ser estudados para avaliar o efeito do levamisol sobre o sistema imune de pacu
Abstract: The emergence of diseases in fish farming is a problem faced in Brazil and worldwide. Some substances can influence the immune system responses of fish, like levamisole, increasing resistance against various etiological agents. Were evaluated the effects of levamisole in diet in two experiments. The first experiment evaluated supplementation with 0, 125, 250 and 500 mg kg-1 imunoestimulante administered by seven and 15 days in which have been assessed the parameters of innate immunity, haematological and biochemists. The administration of levamisole by seven and 15 days promoted changes in haematological and immunological parameters. The administration of levamisole can promote immunodulation, however the determination of the effect not clear due to contradictory answers evaluated in each parameter. The second experiment consisted of supplementation with 0, 125, 250 and 500 mg kg-1 imunoestimulante administered by seven days together with immunization with inactivated Aeromonas hydrophila bacteria with formaldehyde. Immunization and administration of levamisole promoted increase antibody titre, serum bactericidal activity, hematocrit, number of erythrocytes, leucocytes and thrombocytes in pacus totals. Administration of levamisole for seven days and immunization pacus promoted improves some parameters of innate and acquired immunity defense. However other protocols must be studied to assess the effect of levamisole on the immune system of pacu
Doutor

Parallel increases in many inflammatory diseases including atopy over the last 40 years suggest that common environmental changes may be promoting inflammatory immune responses. Modern diets have become increasingly rich in n-6 polyunsaturated fatty acids (PUFAs) and relatively deficient in n-3 PUFAs. These dietary changes are believed to promote a pro-sensitisation, pro-allergic and pro-inflammatory environment. Exposure to such an environment during pregnancy and in the very early life period is considered to influence subsequent patterns of the immature and developing neonatal immune system, and this may contribute to the increase in allergic disease in early life. As allergic diseases often first manifest in infancy, prevention strategies need to be targeted early, even in utero. Epidemiologic and experimental data provide a plausible link between dietary changes and increased incidence of childhood atopic disease. Although there have been studies examining the potential benefits of giving n-3 PUFA-rich fish oil supplements during pregnancy, there are no studies examining the effects of increased consumption of oily fish in pregnancy on neonatal immune responses and subsequent clinical outcomes. The Salmon in Pregnancy Study (SIPS) is the first randomised controlled trial of oily fish intervention during pregnancy. The hypotheses being investigated in SIPS is that increased intake of salmon, a source of long chain (LC) n-3 PUFAs (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)), in pregnancy will a) increase maternal LC n-3 PUFA intake, b) increase maternal and infant blood LC n-3 PUFA status, c) modulate fetal/neonatal immune responses and d) lower the risk of infant atopy determined at 6 months of age. The primary outcome measures of SIPS were the clinical signs of atopy in the offspring. Pregnant women (n=123) at high risk of having atopic offspring, and with low habitual intake of oily fish (≤ 2/month) were randomised at 20 weeks of pregnancy to either consuming 2 portions/week of farmed salmon (n=62) or continuing their habitual diet (n=61) until the end of pregnancy. The woman attended a clinic at 20 (n=123), 34 (n=110) and 38 (n=91) weeks of gestation at which fasting blood was collected and a food frequency questionnaire (FFQ) was administered (at 20 and 34 weeks). At delivery umbilical cord blood was collected (n=101) for fatty acid and immunological analysis. Infants attended a clinic at 6 months of age (n=86) for assessment of allergic sensitisation by skin prick testing (SPT) using various allergen extracts and of atopic dermatitis (SCORAD index). Maternal and cord plasma and cord blood mononuclear cell (CBMC) fatty acid compositions were determined by gas chromatography. Neonatal (cord) immune cell subsets were identified by flow cytometry. Ex-vivo cytokine production by CBMC in response to stimulants (allergen, mitogen, and toll-like receptor (TLR) ligands) was determined by cytometric bead array and flow cytometry. Ex-vivo prostaglandin E2 production by CBMC was determined by enzyme-linked immunosorbent assay. Immunoglobin E concentration was measured in cord blood plasma and in 6 month infant blood plasma. Eating oily fish twice a week during pregnancy resulted in a higher maternal intake of LC n-3 PUFAs (both EPA and DHA) and in higher maternal and cord blood plasma status of LC n-3 PUFAs (both EPA and DHA). LC n-3 PUFA content of CBMC was not significantly affected. CBMC production of interleukins-2, -4, -5, and -10 and tumour necrosis factor-α was lower in the salmon group. There was no effect of salmon on the atopic outcomes assessed at 6 months.

Chez les vertébrés gnathostomes, le système immunitaire repose en grande partie sur les lymphocytes T qui se développent dans un organe conservé évolutivement : le thymus. Chez les mammifères, cet organe constitue une cible privilégiée pour les œstrogènes. La question soulevée ici est donc de savoir si c’est également le cas chez les poissons téléostéens. Dans ce but, la distribution des différents sous-types de récepteurs aux œstrogènes a d’abord été étudiée dans le contexte d’une description de l’anatomie fonctionnelle du microenvironnement thymique. Par la suite, l’expression de gènes relatifs à la fonction thymique et aux différents sous-types de lymphocyte T a été analysée dans le thymus, le rein-antérieur et la rate de bars exposés au 17ß-œstradiol. De plus, la capacité de flambée oxydative a été évaluée sur des leucocytes du rein-antérieur et de rate à la suite d’expositions in vivo et in vitro. Finalement, la variation du nombre de thymocytes a été examinée sur des bars capturés durant trois ans. La thèse fournit de nouvelles connaissances concernant l’évolution des fonctions immunomodulatrices des œstrogènes sur la différenciation des cellules T. En effet, en plus d’une organisation morpho-fonctionnelle fortement conservée, la distribution des sous-types de récepteurs aux œstrogènes ainsi que les effets œstrogéniques apparaissent conservés au cours de l’évolution. Nos résultats suggèrent que, chez le bar comme chez les mammifères, les œstrogènes (1) stimulent une voie alternative de maturation des lymphocytes T ayant des propriétés similaires aux cellules immunitaires innées, (2) augmentent la tolérance immunitaire et (3) régulent la plasticité du thymus
Jawed vertebrates have developed an efficient adaptive immune system partly based on T lymphocytes. They develop in an evolutionarily conserved organ, the thymus. In mammals, endogenous oestrogens are well known to regulate thymus function and plasticity. The question is, therefore, whether this is also the case in lower vertebrates, such as teleosts. To achieve these aims, firstly the distribution of oestrogen receptor subtypes was investigated on the background of a detailed description of the functional anatomy of the thymic microenvironment. Secondly, thymic function- and T cell-related gene expression was analysed in the thymus, the head-kidney and the spleen of sea bass exposed to 17ß-oestradiol. Moreover, the oxidative burst capacity in the two latter organs was evaluated in vivo and in vitro in leucocytes of the head-kidney and spleen following exposure to oestrogen. Eventually, age- and size-dependent variations in thymocyte number were examined in sea bass caught at various time points over three years. The thesis provides new insights into the evolution of the immunomodulatory function of oestrogen with respect to the thymic and peripheral T cell differentiation in vertebrates. As a matter of fact, in addition to a highly conserved morpho-functional organisation, the distribution of oestrogen receptor subtypes as well as the oestrogenic effects appear to be evolutionarily conserved. Our results suggest that in sea bass, similar to mammals, oestrogen (1) stimulates a thymic alternative pathway of T cell maturation with innate-like properties, (2) enhances immune tolerance by promoting Treg differentiation, and (3) actively regulate thymic plasticity

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.

Three investigations were conducted in order to investigate the effect of dietary probiotics on tilapia (Oreochromis niloticus) growth performance, intestinal morphology, intestinal microbiology and immunity. The first experiment demonstrated that Bacillus subtilis, Lactobacillus reuteri and Pediococcus acidilactici supplemented individually and as a mixed probiotic (in addition to Enterococcus faecium; AquaStar® Growout) were capable of modulating intestinal microbial populations as determined by culture dependent methods and DGGE. Furthermore, high-throughput sequencing reported that >99% of 16S rRNA reads in the mixed probiotic group belonged to the probiotic genera, predominantly assigned to Enterococcus (52.50%) and Bacillus (45.94%). Tilapia in the mixed probiotic group displayed significantly higher intraepithelial leucocyte (IEL) populations in the mid intestine when compared to the control and L. reuteri treatment. The mixed probiotic also improved microvilli density and had a higher absorptive surface area when compared to the control. In the second trial, after six weeks of supplementing tilapia diets with AquaStar® Growout at 3g kg-1, fish demonstrated significantly higher final weight, weight gain and SGR when compared to that of the control (void of probiotic) treatment or an initial probiotic feed (lasting two weeks) followed by control feeding. Probiotic supplementation at 3g kg-1 also caused an increase in the abundance of intestinal IELs and goblet cells and an up-regulation in the gene expression of intestinal caspase-3, PCNA and HSP70 and immunity genes TLR2, TNFα, IL-1β, TGFβ and IL-10 when compared with the expression of control replicates. These changes were not observed when supplementing tilapia diets with a lower dose (1.5g kg-1), nor when supplementing the probiotic in either a pulsed manner or as an initial feed (two weeks) followed by control feeding. Trial three revealed that the probiotic had a more discrete effect on the intestinal allochthonous microbiota as 16S rRNA reads assigned to probiotic genera only accounted for 5-10% of total reads. Nevertheless, the supplementation of dietary AquaStar® Growout at 3g kg-1 improved the localised immune response in tilapia, through the regulation of immunity genes TLR2, MYD88, NFκB, TNFα, IL-1β, TGFβ and IL-10, larger populations of goblet cells and a higher recruitment of IELs. Furthermore, the probiotic also improved the systemic immune response through the regulation of immunity genes (mentioned above) in the head kidney and significantly higher circulating leucocyte levels in whole blood. The extent of these changes were dependent on the probiotic treatment (i.e. continuously supplemented in feed or alternating weekly between probiotic at 3 g kg-1 and control feeding), the duration of feeding and the parameter investigated. This research demonstrates that B. subtilis, L. reuteri, P. acidilactici and AquaStar® Growout can modulate the intestinal microbiota. In addition, AquaStar® Growout can improve intestinal morphology, growth performance and modulate both the localised and systemic immune responses of tilapia when supplemented through the feed at the appropriate dosage and feeding regime.

Pela utilização de métodos bioquímicos, biofísicos, de tipagem sorológica e de visualização das proteínas totais bacterianas, isolados de surubim Pseudoplatystoma corruscans, tilápia Oreochromis niloticus e pacu Piaractus mesopotamicus, foram caracterizados, identificados e sua virulência determinada. Dentre as linhagens de referência, o isolado de surubim caracterizou-se como sendo Plesiomonas shiguelloides e os demais isolados de tilápia e pacu foram identificados como Aeromonas hydrophila, todos pertencentes à família Vibrionaceae. Os isolados de tilápia e pacu caracterizaram-se como linhagens virulentas, resistentes aos antibióticos ampicilina, amoxicilina, lincomicina, novobiocina, oxacilina, penicilina, rifampicina e trimetoprim+sulfametoxazol, em ensaios de antibiograma realizados em meio YEA que evidenciaram que as linhagens isoladas de peixes são resistentes a oito das dezessete substâncias antimicrobianas testadas pelo método de difusão em disco. Essas características são compatíveis com as apresentadas pelo espécime tipo de A. hydrophila. Ambas as linhagens quando cultivadas em meio YEA compartilharam a mesma banda de aproximadamente 33,61kDa com o espécime tipo para A. hydrophila. Em meio enriquecido com glucose, a banda compartilhada entre elas teve peso molecular aproximado de 144,28kDa. Os testes de aglutinação sorológica evidenciaram nestas duas linhagens a presença de antígenos estáveis ao calor do tipo O. A técnica de dupla imunodifusão de Ouchterlony demonstrou que o antígeno preparado a partir do isolado de tilápia é a linhagem de A. hydrophila mais indicada para ser utilizada em estudos visando o desenvolvimento de uma vacina polivalente.
Bacteria isolated from surubim Pseudoplatystoma corruscans, tilapia Oreochromis niloticus e pacu Piaractus mesopotamicus, were characterized and identified by biochemical, biophysical, serology, and SDS-PAGE, and their virulence observed. The strain isolated from surubim was characterized as Plesiomonas shigelloides. The other strains isolated from tilapia and pacu were Aeromonas hydrophila. The isolated A. hydrophila strains presented virulence and resistance against the follow antibacterial substances: ampicillin, amoxicillin, lincomicin, novobiocin, oxacillin, penicillin, rifampin and trimetoprim+sulfametoxazole. Both strains when cultivated in YEA medium shared with the A. hydrophila type strain a similar protein band of 33,61kDa. In a medium supplemented with glucose, only one protein exhibiting relative molecular mass of 144.28 kDa, was shared by the type strains isolated from fish and the type strain. The serology tests revealed that all isolated strains presented heat-stable O-antigens. The Ouchterlony double-immunodiffusion showed that the antigen prepared from the tilapia strain possessed surface antigens similar to A. hydrophila type strain and the strains isolated from pacu. This suggested the possibility of development and usage of a common or polyvalent vaccine for A. hydrophila among tilapia and pacu or other freshwater fish species.

Recombinant and DNA vaccines utilizing Mycobacterium sp. antigen 85A (Ag85A) were assessed for immunostimulatory and protective effects against M. marinum. Because of their known susceptibility to piscine mycobacteriosis, Morone sp. were utilized as the models for these studies. The first study evaluated a recombinant vaccine with a Brucella abortus strain RB51 vector expressing the Mycobacterium bovis Ag85A. Striped bass (M. saxatilis) were inoculated at doses equivalent to 106, 107, 108, 109, and 1010 colony-forming units/fish. Vaccinated fish demonstrated significant specific humoral and cell-mediated immune responses towards the Ag85A in a dose-dependant manner. However, vaccinated fish failed to demonstrate cross-protective responses after live Mycobacterium marinum challenge 70 days post-vaccination. A DNA vaccine was constructed utilizing the Mycobacterium marinum Ag85A gene and a commercially-available eukaryotic expression vector. Hybrid striped bass (M. saxatilis x M. chrysops) were immunized by intramuscular (i.m.) and intraperitoneal (i.p.) injection at doses of 5 μg, 25 μg, or 50 μg plasmid. These fish produced significant Ag85A-specific antibody and lymphoproliferative responses over those of control fish injected with saline or empty plasmid. Non-specific macrophage phagocytic and respiratory burst functions failed to exhibit significant upregulation after vaccination. Fish receiving the DNA vaccine developed protective responses to high-dose M. marinum challenge 90 days post-vaccination, as demonstrated by increased relative percent survival and by reduced splenic bacterial counts over control fish. Furthermore specific immunostimulatory and protective effects were significantly increased using higher vaccine doses and using the i.m. injection route. Given these promising findings, the protective responses induced by the DNA vaccine were further investigated. Hybrid striped bass were injected with 25 μg or 50 μg plasmid i.m. and developed specific protective responses to high-dose M. marinum challenge 120 days post-vaccination. The 25 μg and 50 μg groups both developed more rapidly and significantly increased immune responses post-challenge over those of the control groups. The vaccination groups also demonstrated increased survival, reduced splenic bacterial counts, and reduced granuloma formation compared to the control groups. However, though the vaccination groups did not demonstrate the same acute effects post-challenge as the control groups, the vaccination groups ultimately developed increased splenic bacterial counts and granuloma formation, and eventually experienced 100% mortalities. Because piscine mycobacteriosis can affect virtually any species of fish, a vaccine against this disease could be widely beneficial to the aquaculture and ornamental fish industries. The vaccines in these studies exhibited significant immunostimulatory capabilities in Morone sp., but only the DNA vaccine showed promise for conferring protection against M. marinum challenge. Though the DNA vaccine only provided limited protection against high challenge doses, future studies may likely find enhanced protective effects against lower, more natural exposure doses.
Master of Science

One of the greatest challenges in finfish aquaculture is combating losses caused by infectious bacterial diseases, and a better understanding of the interactions between the host immune system and pathogens is essential for developing new methods to manage infections and outbreaks. Extracellular traps (ETs) are decondensed nuclear chromatin released by neutrophils into the extracellular matrix that can ensnare and kill microbes. Since the discovery of ETs in humans, these innate immune effectors have been characterised across the animal kingdom, including in some fish species, though their existence the salmonids has yet to be confirmed. Therefore, the aim of this thesis was to confirm and characterise the release of ETs in the rainbow trout (Oncorhynchus mykiss) and investigate the interaction of these structures with fish pathogenic bacteria. To do this, a triple-layer Percoll gradient technique was employed to give highly enriched cell suspensions of polymorphonuclear cells (PMNs) derived from head-kidney tissue preparations. Treatment of PMN-enriched cell suspensions with the nucleic-acid-specific stain, SYTOX Green, revealed the presence of ET-like structures that had been released without stimulation. These ET-like structures were confirmed by immunostaining techniques to contain the diagnostic proteinaceous markers of ETs: neutrophil elastase, myeloperoxidase and the H2A histone. Previously characterised inhibitors and inducers of ET release from phagocytic immune cells in other animals confirmed that calcium ionophore (CaI), flagellin, and cytochalasin D shared similar activities for ET-release by rainbow trout PMNs. However, interestingly, as the common ET-inducer phorbol-myristate acetate (PMA) and ET-inhibitor diphenyleneiodonium (DPI) did not exert their expected potency in ET release assays with the PMNs, perhaps indicating that these fish cells are less dependent on NADPH oxidase signalling for ET release compared to mammals and most invertebrate species. The PMN-derived ETs were demonstrated to bind to and trap the extracellular nuclease-deficient bacterial fish pathogen, Vibrio anguillarum (Vib 87) when co-cultured. Finally, extracellular nuclease activity produced by a V. anguillarum isolate (Vib 6) during culture was able to degrade ETs released by rainbow trout PMNs in a dose-dependent manner. Moreover, viable colony counts, fluorescent and phase contrast microscopy demonstrated that V. anguillarum Vib 6 eluded trapping by ETs, while an extracellular nuclease-deficient isolate did not. These observations are consistent with the suggestion that nucleases are a microbial virulence factor during host infection. Confirming the existence and antimicrobial potential of extracellular traps released by rainbow trout PMNs may provide a platform towards the development of novel therapeutics to reduce mortalities in finfish aquaculture caused by infectious microbial pathogens.

L’objectif principal de cette thèse est d’étudier les mécanismes liés au pouvoir pathogène de V. tapetis.Pour cela, nous avons développé 2 axes de recherche. Le premier axe vise à étudier la virulence de V. tapetis en répondant aux 2 problématiques suivantes : Quels sont les gènes impliqués dans la virulence de V. tapetis ? et Existe-t-il des marqueurs hôtes-spécifiques de la virulence de V. tapetis ? Le second axe de recherche concerne l’interaction hôte pathogène et répond aux 2 problématiques suivantes : Quels sont les gènes exprimés lors de l’infection chez l’hôte ? et Quelles sont les modulations au sein de l’animal associées au pH et à la température lors de l’infection ?Les principales découvertes de cette thèse sont : (i) La bactérie V. tapetis, dans le cadre de la MAB, induit une sous expression des gènes impliqués dans la réponseimmunitaire et une dérégulation des gènes impliqués dans la stabilisation et la synthèse des filaments d’actine (ii) Ce pathogène induit également une diminution de l’activité lysosomale sur les hémocytes exposés (iii) L’effet de V. tapetis sur le cytosquelette d’actine et sur la diminution de l’activité lysosomale est indépendante du système de sécrétion de type IV (T4SS) (iv) Le système de sécrétion de type IV (T4SS) est impliqué dans le développement de la MAB mais n’est pas essentiel pour induire cette affection(v) Dans le cadre de la MAB et de la perte des adhérences des hémocytes in vitro, V. tapetis est capable de moduler le pH des fluides extra-palléaux, respectivement dans les premiers jours et premières heures de l’infection (vi) Enfin, l’approche de « strains typing » basée sur la technique MALDI-TOF permet de discriminer les souches de V. tapetis en fonction de leur pouvoir pathogène vis à vis de la palourde japonaise
The main objective of this thesis is to study the mechanisms related to the pathogenicity of V. tapetis. For this purpose, we developed 2 research axes. The first one aimed at studying the virulence of V. tapetis by answering the following 2 issues: What are the genes involved in the virulence of V. tapetis? and Are there host-specific markers of the virulence of V. tapetis? The second research axis concerned pathogen-host interactions and addressed the following 2 issues: What are the genes expressed during infection in the host? and What are the modulations in the animal associated with pH and temperature during infection? The main findings of this thesis are: (i) V. tapetis, in the context of BRD, induces a down expression of genes involved in the immune response anda deregulation of genes involved in the stabilization and synthesis of actin filaments (ii) This pathogen also induces a decrease in lysosomal activity on exposed hemocytes (iii) The effect of V. tapetis on the actin cytoskeleton and on the decrease in lysosomal activity is independent of the type IV secretion system (T4SS) (iv) The type IV secretion system (T4SS) is involved in the development of BRD but is not essential to induce this disease (v) In the context of BRD and of the loss of hemocyte adhesions properties in vitro, V. tapetis is able to modulate the pH of extrapallial fluids, respectively in the first days and hours of infection (vi) Finally, the "strains typing" approach based on MALDITOF makes it possible to discriminate between V. tapetis strains according to their pathogenicity with regard to Manila clam

A contribuição de células B para a memória imunológica se dá por duas distintas populações: células B de memória e células produtoras de anticorpos de longa vida (ASC). A inter-relação entre estas células bem como os mecanismos envolvidos para a manutenção destas tem sido pouco entendida. O veneno de Thalassophryne nattereri tem se mostrado capaz de induzir uma intensa resposta imune de memória. Nós avaliamos o efeito das Natterinas, que são as toxinas majoritárias e inéditas, na indução e manutenção da resposta imune de memória de células B. Este estudo, além de permitir um maior esclarecimento da resposta humoral de memória induzida pelo veneno do peixe T. nattereri, permitiu o estudo da complexa organização do compartimento de células B de memória e ASCs. Também evidenciamos a importância da atividade proteásica para a manutenção da cronicidade de resposta de células B no peritônio, no baço e na medula, como verificamos que a ativação de receptores inatos como osTLRs é decisiva para a geração e manutenção de ASCs B220pos/neg em resposta às Natterinas, dependentes das vias de sinalização MyD88 ou TRIF. Estas sinalizações controlam a magnitude, a qualidade e a longa duração da resposta humoral de memória.
The contribution of B cells for the immunological memory feels for two different populations: memory B cells and long-lived antibodies secreting cells (ASC). The interrelation among these cells as well as the mechanisms involved for the maintenance of these it has been little understood. The venom of Thalassophryne nattereri possesses the ability to induce an intense memory immune response. We evaluated the effect of Natterins that are majority toxins in the venom, in the induction and maintenance of the immune memory response of cells B. The study, besides allowing a larger explanation of the humoral memory response induced by the venom of the fish, it allowed the understanding of the complex organization of the memory B cells compartment, mainly of the subtype of long-lived cells (ASC). Also, we showed the importance of the protease activity of Natterins in the maintenance of the chronic B cell responses in the three analyzed compartments. We verify that the activation of Toll like receptors is decisive for the generation and maintenance of ASCs B220pos/neg in response to Natterins, dependent on the MyD88 or TRIF signaling that control the quality and the duration of the humoral memory response.

Dans le cadre de l’évaluation du risque immunotoxique de composés glycolypidiques d’origine bactérienne, cette thèse a portée sur l’évaluation de toxicité d’endotoxines d’E.coli de deux sérotype différents : LPS O55:B5 utilisé couramment en comme immunostimulant et le LPS O157:H7 dont la réalité environnementale a soulevé notre questionnement scientifique quant à son impact sur le système immunitaire du poisson et son caractère potentiellement pro-inflammatoire. Les différents procédés employés ont compris l’évaluation des paramètres cellulaires (production d’espèces réactives d’oxygène et phagocytose) ainsi que la caractérisation et la quantification de l’expression de gènes de cytokines (TGFβ et IL-10) et facteurs immuno-associés (MARCO, HSP60 et vitellogénine) chez le modèle gardon (Rutilus rutilus).Les approches expérimentales se sont déroulées tout d’abord en ex vivo sur des leucocytes isolés d’organes lymphoïdes (Rein antérieur, rate et sang) de gardon et ont montré une tolérance endotoxique vis-à-vis de ces LPS à de 1µg/mL même combinés au diclofénac à 0,1 µM. ce travaill a été suivi par une évaluation du risque potentiel d’autres composés glycolipidiques d’origine bactérienne (rhamnolipides).Les approches in vivo qui ont suivies ont été réalisées sur : (i) sur modèle poisson-zèbre (Danio rerio) au laboratoire et (ii) sur modèle gardon par encagement sur terrain. Les résultats obtenus sur danios au laboratoire ont montré une toxicité du sérotype O157 :H7 et une influence sur les paramètres comportementaux par les LPS (Sickness behaviour). Sur terrain, l’approche in vivo par encagement a révélé – au niveau de la rate et du rein antérieur – des réponses cellulaires et moléculaires, sérotype, organe et sexe dépendante avec une immunomodulation prédominante chez les mâles d’autant plus que la période d’étude s’est déroulée durant la maturation sexuelle des gardons. Ce travail fait état du caractère inflammatoire et toxique du LPS peu étudié d’E.coli O157 :H7, évalué par des immunomarqueurs cellulaires bien maitrisés et moléculaires néo-développés
In the context of immunotoxic risk evaluation of glycolypidic compounds of bacterial origin, this thesis focused on the evaluation of E.coli endotoxin toxicity of two different serotypes: LPS O55: B5 commonly used as an immunostimulant et LPS O157: H7, whose environmental reality has raised our scientific questioning about its impact on the fish's immune system et its potentially pro-inflammatory nature. The various methods used included the evaluation of cellular parameters (production of reactive oxygen species et phagocytosis) as well as the characterization of cytokines (TGFβ et IL-10) et immune-realted factors (MARCO, HSP60 et vitellogenin) genes et the quantification of their expression in the roach model (Rutilus rutilus).The experimental approaches were first carried out ex vivo on leukocytes isolated from lymphoid organs (anterior kidney, spleen et blood) roach et showed an endotoxic tolerance at 1μg / mL even combined with 0.1 μM diclofenac. This work was followed by an evaluation of the potential risk of other glycolipidic compounds of bacterial origin (rhamnolipids).The in vivo approaches that followed were performed on: (i) zebrafish (Danio rerio) model in the laboratory et (ii) on roach model by field caging. The results obtained on danios in the laboratory showed a toxicity of the serotype O157: H7 et an influence on the behavioral parameters by the LPS (Sickness behavior). On the field, the caging approach revealed - at spleen et the anterior kidney level - cellular et molecular responses, serotype, organ et sex-dependent with a predominant immunomodulation in males, especially since the study period took place during the sexual maturation of roaches. This work reports the inflammatory et toxic nature of the less studied E.coli O157: H7 LPS serotype, evaluated by well-mastered cellular et neo-developed molecular immunomarkers

Chemokines are chemotactic cytokines that have the ability to attract leukocytes and guide them via a concentration gradient to sites of injury/infection. These small, basic proteins are secreted when induced and act on their target cells through G-protein coupled receptors. The downstream effects of this family of immune molecules are vast and have not fully been characterized. These versatile molecules seem to be able to serve as a link between the innate and adaptive immune systems as they are capable of inducing the inflammatory response of the innate immune system as well as triggering the initiation of the adaptive immune system. There are currently 46 known chemokines in humans. Chemokines have also been isolated from mouse, chicken, frog and fish. The rainbow trout chemokine CK-2 is the only known functional CC chemokine in possession of a mucin stalk. A previous study in this lab showed that stimulation by PHA causes a decrease in CK-2 transcript levels in rainbow trout head kidney and peripheral blood leukocytes (PBLs) as well as in the rainbow trout macrophage-like cell line RTS-11. CK-2 protein was found expressed in RTS-11 but not in the spleen tissues of stimulated fish. A chemotaxis assay was performed to determine the activity of recombinant CK-2. It was observed that recombinant CK-2 induces the migration of rainbow trout PBLs as well as RTS-11 cells at an optimal concentration of 100ng/mL when purified under native conditions. The migration of cells treated with pertussis toxin is significantly reduced, indicating that it relies on G-protein coupled receptors. Treatment of RTS-11 cells with recombinant CK-2 results in changes in the expression profiles of various immune response genes including those that are involved in the inflammatory response and the responses against both intracellular and extracellular pathogens. Interestingly, rCK-2 induced an upregulation in the expression of the surface molecule CD4 at the level of transcription. The increase in CD4 may suggest a possible role for CD4 in the regulation of the cell’s response to chemokines, indicating a potential function for the molecule in macrophages which has yet to be determined. This study shows that CK-2 is a functional chemokine that has a role in the rainbow trout immune response involving, but not limited to, macrophages.

Little is known about the virulence factors of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. The predominant protein produced by R. salmoninarum in broth culture or during infection is a 57/58 kDa protein (p57) which is associated with strain virulence. In this study monoclonal antibodies (MAbs) to p57 were developed and used as tools to antigenically characterize and quantify the protein. Monoclonal antibodies 4D3 and 2G5 recognize p57 and appear to be species specific as they did not cross-react with proteins produced by bacterial species within the genera Streptococcus, Carnobacterium, Vibrio and Aeromonas, or with fish serum proteins. Further, these MAbs recognize conserved epitopes on p57 shared by 10 isolates from geographically diverse areas. In vitro activities attributed to p57 include the suppression of antibody production, and the agglutination of rabbit erythrocytes and salmonid spermatocytes. We described a novel in vitro agglutinating activity of p57 toward salmonid leukocytes that was inhibited by two of a panel of eight MAbs. The location of the putative epitopes recognized by the MAbs were determined by two-dimensional electrophoresis and Western blotting of proteolytic breakdown fragments of p57. Amino acid sequencing of several of the fragments suggested that the antibodies which inhibit agglutinating activity bind proximal to the amino terminus of the protein. To investigate the mechanism of leukocyte agglutination, p57 was purified to near homogeneity using anion-exchange and size-exclusion fastpressure liquid chromatography. P57 eluted as a protein monomer and retained leukoagglutinating activity. In addition, results of antibody-capture, enzyme-linked immunosorbent assays suggest that a monomer exists in culture supernatant and infected fish tissue. Antigenic analysis with MAbs has also been useful for developing immunoassays for detecting and quantifying p57 levels in vivo. Using a quantitative ELISA, the prevalence of salmon with antigen levels above 3 ng/ml of kidney homogenate varied from 12.8 to 36.6% in 740 adult spawning chinook salmon returning to an Oregon hatchery from 1989 to 1991. A rapid, semi-quantitative, Field ELISA was also developed for use under hatchery conditions, in addition to a sensitive chemiluminescent Western blot protocol for confirming ELISA positive samples.
Graduation date: 1992

Stressors in the aquaculture environment can lead to negative impacts on growth and immune health, resulting in susceptibility to infectious diseases. These stressors are expected to increase as the growth of aquaculture continues to rise to meet demands for quality fish protein. Isoflavones, as a crude extract or as a pure isolate, may be effective in modulating the stress response, promoting growth and immunity. The objective of these studies was to examine the effect of various pure isoflavone isolates and crude isoflavone extracts on stress, growth, and immunity. Nile tilapia (Oreochromis niloticus) were stressed by adding hydrocortisone to the feed. In a 7-week study, pure isoflavone isolates of genistein and puerarin were evaluated to determine their respective effects on stress, growth, and immunity. A separate 10-day physiological and 6-week growth study focused on crude isoflavone extracts from kudzu (Pueraria lobata), red clover (Trifolium pratense), and soybean (Glycine max) was performed to determine their respective effects on stress, growth, and immunity. Numerous physiological parameters of the fish were measured (serum cortisol concentration, blood glucose concentration, hematocrit, hepatosomatic index, plasma protein concentration, lysozyme activity, and spleensomatic
index) to determine the effects of these pure isoflavone and crude isoflavone extracts on the modulation of stress and immunity. Many growth parameters were examined (length, weight, condition factor, weight gain, specific growth rate, feed intake, feed conversion ratio, and protein efficiency ratio) as well to determine the effects of these pure isoflavones and isoflavone extracts on growth. The addition of isoflavone and crude isoflavone extracts to the diet of Nile tilapia ameliorated some of the negative consequences of stress. Compared to stressed fish fed commercial feed, genistein and puerarin added to the diet appeared to improve serum cortisol concentrations, which resulted in increased plasma protein, albeit at different durations of stress. Puerarin, as well as all three crude isoflavone extracts, significantly increased spleen-somatic index compared to non-supplemented stressed fish, although the crude isoflavone extracts did not appear to improve serum cortisol concentrations. Crude isoflavone extracts also showed overall increases in lysozyme activity compared to non-supplemented stressed fish, although this was not significant. Genistein, puerarin, and red clover showed increased growth rates, feed conversion ratio, and protein efficiency. Overall, pure isolates of isoflavone appear to be more effective in modulating stress, immunity, and growth than the crude isoflavone extracts, although red clover extract showed promises in the ability to modulate the stress response and improve growth and immunity. There are likely substantial interactions between the isoflavones in the crude extracts that cannot be fully understood by measuring the effects of single isoflavones. Regardless, isoflavone supplementation (pure or crude) appeared to generally have an overall positive impact on stressed Nile tilapia, requiring more research to better understand the effects and mechanisms behind these isoflavones.