A series of investigations were conducted in order to characterise the GIT microbiota of salmonids and to determine the effect of microbial modulating feed additives on the intestinal microbiota, immunity and growth of salmonids. The first experiment, Chapter three, used PCR-DGGE and 16S rRNA gene sequence analysis of cultivable bacteria were used to investigate the GIT microbiota of brown trout. 16S rRNA gene sequence analysis demonstrated that Citrobacter freundii and Carnobacterium maltaromaticum were the predominant culturable viable bacteria and lactic acid bacteria, respectively in all regions of the GIT. DGGE revealed complex communities with a diverse range of microbes from the Firmicutes and Proteobacteria phyla. The latter chapters focused not only identifying the gut microbiota of salmonids, but also on the ability of probiotics and prebiotics to modulate these communities. In Chapter four, rainbow trout were fed a commercial diet supplemented with P. acidilactici for four weeks. P. acidilactici was detected in the GIT of the probiotic group by multiple methods and P. acidilactici was able to persist for at least 24h at the cessation of probiotic feeding. Histological appraisal on the intestine revealed significantly higher microvilli density in the posterior mucosa and a higher density of goblet cells in the anterior mucosa of the probiotic fed fish. RT-PCR results demonstrated that IL-1β, IL-8 and IgT gene expression were up-regulated in the P. acidilactici fed fish at the end of the study. Whilst mRNA of PCNA, HSP70 and casp-3 were down-regulated in the probiotic group at both sampling points. In Chapter five, the efficacy of dietary administration of P. acidilactici and short chain fructooligosaccharide (scFOS) on Atlantic salmon (Salmo salar L.) was evaluated at 63 and 132 days. Compared to the control group, total bacterial cell counts in all regions of the intestine with exception of the anterior digesta were significantly lower in the synbiotic group at the mid sampling point. PCR-DGGE revealed that species richness, diversity and the number of OTUs were significantly higher in the synbiotic group in the anterior digesta at the mid sampling point. Intestinal microvilli and villi length were increased in the anterior intestine of the synbiotic fed group at the end sampling point. IEL levels were increased in the synbiotic group in the posterior intestine at both sampling points. The expression of immunological genes were significantly up-regulated in the synbiotic fed salmon. In Chapter six, rainbow trout were fed three diets fishmeal (FM), soybean meal (SBM) and PlantMix diets supplemented with or without P. acidilactici for 12 weeks. At both sampling points, with exception of fish fed FM, LAB levels were significantly higher in all probiotic groups compared to the control groups. Serum lysozyme activity was significantly higher in fish fed FM and SBM diets containing P. acidilactici than that of fish fed the control diets. This body of research demonstrates that P. acidilactici can modulate immune response via up-regulation of immune genes as well as modulate IEL and goblet cell levels. Despite these benefits, P. acidilactici had no detrimental effects on growth performance.

Aquaculture is being seriously considered as a promising sustainable industry in the Sultanate of Oman. Fish farming commenced in Oman in 1986, but it was only in 2011 that it became a more commercially driven sector. While worldwide aquaculture production is expected to rise to meet the shortage in capture fisheries, there is a parallel requirement to identify potential threats to the health and welfare of existing aquatic farmed stocks and to take appropriate steps to mitigate them. As aquaculture in Oman is in an early stage of development, it is important to acquire baseline data on the existence and prevalence of aquatic diseases and pathogens to help the Government make policy decisions to develop health management regimes applicable for Omani aquaculture. Therefore, this study was conducted to evaluate current farming practices of tilapia in Oman, to investigate the bacterial species composition and distribution from different sites in some of the economically important fish species, and to study the characteristics and pathogenicity of Aeromonas species. The current practices were studied for 9 Nile tilapia (Oreochromis niloticus) farms from four areas (Al Batinah, Ad Dhahirah, Ad Dakhiliyah and Ash Sharqiyah North) during the period of September to November 2012 by using questionnaires and interviews with the farm owners and staff. In total 417 fish representing 5 target species were chosen on the basis of the commercial importance and their potential for aquaculture in Oman, including red spot emperor (Lethrinus lentjan), king soldier bream (Argyrops spinifer), white spotted rabbit fish (Siganus canaliculatus), abalone (Haliotis mariae) and tilapia (Oreochromis niloticus). The fish were collected from 5 main sampling areas in Oman (Muscat, Mudhaibi, Manah, Sohar and Salalah) based on the Atlas of suitable sites for aquaculture in Oman to investigate the bacterial species composition and distribution. The animals were examined for clinical signs of disease prior to routine bacteriology. Bacterial isolates were recovered using traditional methods and identified to species level using phenotypic and molecular approaches using 16S rDNA, 16S rDNA RFLP and 16S rDNA sequencing. Experimental fish challenge studies were also conducted using both live bacterial cells and ECP protein to investigate the pathogenicity of Aeromonas isolates. In addition, the presence of some virulence factors was investigated using both phenotypic and genotypic methods. The results of this study showed that, the most farms in the Oman follow very similar farming practices. The major proportion of the tilapia is consumed within the local communities. A number of farmers have experienced mortalities, which were considered to be attributable to poor water quality, overcrowding or due to excessive feeding. Farmers facing fish mortalities tended not to record the problems due to a lack of understanding of the concept of fish farm management. There is a regulation about aquaculture and related quality control, but it has not yet been implemented in an appropriate manner in Oman. From the diverse group of bacteria recovered from wild and farmed fish, 83% of the total isolates comprised Gram negative, rod-shaped bacteria. The most frequently isolated groups from marine and cultured fish were Aeromonas spp., Vibrio spp., Sphingobacterium spp., Micrococcus spp. and Staphylococcus spp., with Aeromonas spp. being the predominant group representing 25% of the isolates recovered in this study. Identification of the Aeromonas spp. showed 57% agreement between the results of phenotypic and genotypic methodologies, and determined 6 species as the dominant organisms, i.e. A. veronii, A. jandaei, A. caviae, A. trota, A. encheleia and A. salmonicida. 65% of the iso-lates shared 99% 16S rDNA sequence similarity with the closest sequences in GenBank, and the dominant species was A. veronii. In conclusion, the Aeromonas isolates recovered from fish with clinical signs of disease showed heterogeneity in their identification profiles and their pathogenicity.

Through the implementation of in vivo feeding trials, the efficacy of three wheat gluten (WG) products, vital (Amytex®), hydrolysed (Merripro®) and soluble hydrolysed (Solpro®) wheat gluten as replacement of soy protein concentrate, and scFOS prebiotic (Profeed®) supplementation were analysed to assess their impacts on intestinal health and production of juvenile rainbow trout. Microbial community analysis in experiment one revealed a degree of diet based modulation with 7.5% and 15% inclusions of wheat gluten (WG) products. Bacterial species diversity was significantly reduced with 15% hydrolysed wheat gluten (HWG) inclusion compared to the plant protein control and 15% vital wheat gluten (VWG) treatments, with sequenced OTUs dominated by the phylum Firmicutes and possible promotion of probiotic species. No detrimental effects were observed on intestinal morphology. These findings led onto a longer duration feed trial with a more holistic, higher resolution approach. Experiment two revealed modulation of the allochthonous intestinal microbiota, with increased proportions of Enterococcus and Weissella in the 10% and 20% VWG treatments. Bacillus and Leuconostoc relative abundances were significantly increased with 10% HWG and soluble hydrolysed (Sol) wheat gluten inclusions. HSP 70 transcripts were significantly down-regulated in all WG treatments compared to the basal soy protein concentrate treatment (SPC) and increased intraepithelial leukocyte counts were observed with 10% VWG inclusion. Growth performance was unaffected by 10% dietary inclusions of WG, however, FCR’s were significantly improved in the 20% VWG treatment compared to the 10% HWG and Soluble treatments. This led to the investigation of increased inclusion levels of WG products in experiment three. All WG treatments in experiment three yielded significantly improved growth performance. Somatic indices were significantly increased with 30% blended WG inclusion compared to the SPC treatment. Modulation of allochthonous intestinal microbiota was observed to a lower degree than the previous experiments, with a dose response observed with increasing blended WG inclusion. In the final experiment two basal diets (SPC and 20% Blended) and two scFOS supplemented diets (SPC + FOS and 20% Blended + FOS) were investigated for the effect on growth performance, gut health and allochthonous microbial population. Growth performance was unaffected, however, modulation of the allochthonous microbial population was observed with an apparent synergistic effect of scFOS supplementation in WG diets. This synergistic trend was also observed in the transcription level expression of immune relevant genes. 20% WG inclusion with additional scFOS supplementation observed significant down regulation of the pro-inflammatory cytokine TNF-α, as well as HSP 70, CASP 3 and Glute ST compared to the 20% Blend treatment. The present research demonstrates dietary inclusions of WG products, solely or blended, at the expense of soy protein concentrate to modulate the allochthonous microbial population, potentially promoting probiotic species, whilst reducing the levels of intestinal stress in juvenile rainbow trout. Supplementation of the prebiotic scFOS modulated the microbial populations, enhancing the proportion of potential probiotic species, and combined with WG inclusions, reduce intestinal and oxidative stress and inflammation biomarkers, with no observed deleterious effects.

In Thailand, culture and production of a high value freshwater fish, the marble goby, is dependant upon farm-made feeds using marine and freshwater trash fish as primary ingredients. However, there is lack of nutritional research regarding the use of such farm-made feeds and their impacts on the nutritional status, growth and health of marble goby. The aims of the present study were to evaluate the effects of farm- made feeds on slaughter indices, fish lipid classes and fatty acid profiles, nutrient composition and digestibility. In addition it was intended to improve on-farm feed quality for both current practical feeds as used by farmers and alternative feeds using rice bran and tilapia with reference to biochemical composition, growth performance and haematology of marble goby. Nutritional evaluation of farmed fish compared to their wild counterparts indicated that fatty acid composition of farmed marble goby was markedly influenced by diet. Marble goby appeared to utilize MUFA preferentially as an energy source compared to SFA. Fish muscle was characterised by higher n-6 PUFA; arachidonic acid (20:4n-6, AA) and docosapentaenoic acid (22:5n-6, DPA). Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22: 6n-3, DHA) comprised the majority of n-3 PUFA found in fish muscle and can potentially be enriched into marble goby muscle through the diet. The significantly higher ratios of neutral liver lipid to polar lipid (NL/PL) indicated the imbalance of dietary lipid and energy of on-farm feeds. Moreover, findings on slaughter indices and lipid peroxidation of farmed fish; higher HSI, VSI, liver TBARS and the pale lipid-rich liver of farmed fish indicated problems with the nutritional quality of lipid in farm-made feeds. Experiments aimed to improve farm-made feeds using supplemental vitamin E in the form of α-tocopherol. These demonstrated that supplementation of α-tocopherol to oxidised diets, both mackerel and tilapia based, did not result in a significant beneficial effect in reducing mortality, and improving growth and haematology in marble goby. However, dietary α-tocopherol supplementation helped in reducing fish muscle peroxidation but was not related to muscle α-tocopherol levels. In mackerel based diets containing lipid peroxidation up to 250-300 µmols MDA g-1, α-tocopherol supplementation appeared not to help in reducing liver peroxidation. The alternative use of tilapia as a feed for marble goby resulted in growth and survival rates similar to those of fish fed mackerel based diets. Tilapia contained intrinsic α-tocopherol levels that appeared to be sufficient to reduce marble goby tissue peroxidation. The synergic effects on antioxidant activities between α- tocopherol supplement and natural E vitamer contained in rice bran helped to reduce tissue TBARS and improve haematology in fish fed combination diets of oxidised tilapia and rice bran. The inclusion of rice bran in farm based diets resulted in decreased tissue peroxidation, an adverse affect on dry matter and protein digestibility; and lower fish feed intake, growth and survival rate when 25% of rice bran was added into practical mackerel based diet. Overall, formulated feeds showed promising growth and survival rate in marble goby but more research on dietary nutrients and energy balances are required.

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.

Three investigations were conducted to assess the effects of selected bio-active feed additives on tilapia (Oreochromis niloticus) growth performance, feed utilisation, haemato-immunological status, intestinal morphology and microbiology. The first experiment was conducted to evaluate the effects of feeding tilapia semi-practical diets supplemented with exogenous enzymes (phytase, protease or carbohydrase). After six weeks of the dietary supplementation, tilapia fed diets supplemented with exogenous enzymes (phytase and carbohydrase) showed enhanced growth performance and higher microvilli density when compared to the control group. Additionally, the intestinal bacterial community profile of tilapia fed the carbohydrase supplemented diet was significantly altered in contrast to those fed the control diet. In the second experiment, tilapia were fed with practical diets supplemented with exogenous enzymes (phytase, protease or xylanase) for eight weeks. Tilapia fed the xylanase supplemented diet demonstrated significantly higher final body weight (FBW), improved feed conversion ratio (FCR) and better protein efficiency ratio (PER) when compared to the control group. Apparent digestibility coefficients of protein, ash, energy, phosphorus, calcium and sodium were highest in tilapia fed a diet supplemented with phytase. The third experiment was carried out to assess the combined effects of dietary exogenous enzymes (phytase, protease and xylanase) and probiotics (Bacillus subtilis, B. licheniformis and B. pumilus) on tilapia growth performance and health. After seven weeks of feeding, tilapia fed the diet supplemented with both exogenous enzymes and probiotics showed significantly higher FBW, specific growth rate, improved FCR and better PER. The serum lysozyme activity was observed to be significantly higher in tilapia fed the probiotic supplemented diet when compared to the control group. The dietary supplementation with combined exogenous enzymes and probiotics increased intestinal perimeter ratio, microvilli count (density), diameter and subsequently resulted in higher enterocyte absorptive area in tilapia. This study demonstrates that feeding tilapia with dietary exogenous enzymes can enhance growth performance and modulate microbial community profile. In addition, supplementation with both exogenous enzymes and probiotics is capable of improving tilapia growth performance, immune parameters and intestinal morphology.

In Sachsen gewinnt die Aufzucht von Stören in Teichen zunehmend an Bedeutung, insbesondere um die Wirtschaftlichkeit der Warmwasserteichwirtschaft zu verbessern. Mit der Störerzeugung wurden Kapazitäten für die Herstellung von Farmkaviar geschaffen. Dieser ist jedoch starken Qualitätsunterschieden unterworfen. Störe neigen zu einer starken Verfettung des Eingeweidekomplexes, des Filets und der Gonaden, was geschmackliche Probleme zur Folge hat. Es wurde untersucht, ob spezielle Futtermittel, die für die finale Aufzuchtphase von Stören angeboten werden, geeignet sind, die Kaviarqualität zu verbessern und der Verfettung entgegenzuwirken. Mit allen vier geprüften Futtermitteln konnten gute Zuwachsleistungen und eine Verbesserung der Produktqualität erreicht werden. Alle Futtermittel erwiesen sich als geeignet für die Störaufzucht in der finalen Aufzuchtsaison vor der Kaviargewinnung. Die eingesetzten Futtermittel hatten jedoch weder Einfluss auf die zu gewinnende Menge, noch auf die Qualität des Kaviars.

Der Einsatz von Branntkalk war noch vor wenigen Jahren Bestandteil der »guten fachlichen Praxis« in der Karpfenteichwirtschaft. Im Hinblick auf den Schutz von Tier- und Pflanzenarten sowie geschützte Biotope wurde der Branntkalkeinsatz seit einigen Jahren vorsorglich eingeschränkt. Der Bericht dokumentiert die Ergebnisse einer Untersuchung zur Wirkung unterschiedlich hoher Branntkalkgaben auf die Wirbellosenfauna, Amphibien, Libellen und Makrophyten in Praxisteichen sowie in der Lehr- und Versuchsteichanlage Königswartha. In Laborversuchen wurden auch die Auswirkungen unterschiedlicher Branntkalkmengen auf Amphibienlaich und -larven untersucht. Die schädigende Wirkung von Branntkalk ist abhängig von der Dosierung. Während Einzelgaben unter 500 kg/ha praktisch ohne Auswirkungen bleiben, sind bei Desinfektionskalkungen auch Artenschutzbelange zu beachten.

Aquaculture production is increasing annualy and wild fisheries for fishmeal production remain stagnant. As a consequence, extensive research has been deployed to reduce dietary fishmeal inclusion in feeds of farmed species. Usage of alternative protein sources derived from plants continues to increase with the most popular sources being oilseeds, legumes and cereal grains. The downside of these sources is that most of them could directly be used for human consumption arising legitimate criticism from voices referring to countries where protein shortages lead to malnutrition among the population. Jatropha curcas is a tropical oilseed with upcoming popularity for sustainable fuel sourcing. The plant is thought to thrive in semi-arid and arid areas, not just producing oil, but at the same time reclaiming previously eroded land for the local population. For these reasons, annual cultivation of Jatropha curcas is thought to rise over the next decades. After oil is extracted from the seed, the remaining press cake, is currently used as a fertilizer or energy source. This is mostly due to toxic phorbol esters that until recent- ly limited any nutritional applications. In 2011, a method to detoxify Jatropha press cake was developed and paved the way for nutritional research on the resulting detoxified Jatropha curcas kernel meal (JKM) to be launched. JKM offers very high protein content with a balanced amino acid composition suggesting opportunities for usage as a feedstuff in aquaculture diets. JKM further has higher mineral content than comparable oilseeds. However, potential anti-nutritional factors (ANFs) present in the meal could lead to impairment of nutrient availability or other adverse effects. Previous research has already started to evaluate JKM as a protein source for a variety of aquaculture species. This thesis attempts to further identify the potential of JKM as a protein source and assess the effects of JKM on the development of two model cultured teleost species, common carp (Cyprinus carpio) and Nile tilapia (Oreochromis niloticus), as they represent two of the most farmed species, particularly in the tropics. The work comprises growth trials on both species using fishmeal as a subject of replacement in solely fishmeal protein-based diets and on more practical fishmeal/plant protein-based diets. It engages with the effects of phytate, a prominent component of JKM with potential anti-nutritional attributes and commercially available phytase feed supplements to diminish these attributes, as well as oxalate, another plant-specific component with high concentrations in JKM and with limited attention in aquaculture nutrition research. Carp and tilapia showed varying results with different inclusion levels of JKM. For carp 50% fishmeal replacement was possible without losses in growth in diets where fishmeal was the only bulk protein source (Chapter 3.1), Tilapia showed slightly worse growth at a 30% replacement level (Chapter 3.2). A steep decline in growth could be observed when replacing 100% fishmeal with JKM in carp (Chapter 3.1), while tilapia showed no difference at that level compared to 30% replacement (Chapter 3.2). In practical diets, 100% of fishmeal could be replaced by JKM without any adverse effects on growth of carp (Chapter 3.3), while tilapia showed a slight, but significant linear negative correlation with higher inclusion levels of JKM (Chapter 3.4). Phytase addition in tilapia feeds was identified as having no obvious impact on growth in JKM based diets where enough available phosphorus was provided through mineral supplementation (Chapter 4.1). In JKM based diets where available phosphate was not added, phytase addition showed a tendency to increase growth and significantly increased mineral retention and decreased phosphorus effluent contamination (Chapter 4.2). Phytase application through pre-incubating JKM along with citric acid exerted a positive effect of growth on carp when fishmeal protein was replaced by 50% (Chapter 3.1). Phytase was further shown to completely hydrolyze phytate in vitro; however, incomplete hydrolysis was observed in vivo in tilapia (Chapter 4.3). Dietary soluble oxalate added to fishmeal based diets for carp showed better growth parameters, nutrient and mineral retention at inclusion levels 1.5% and higher (Chapter 5.1). For tilapia, a trial could demonstrate adverse effects of oxalate on potassium, calcium, manganese and zinc digestibilities, in this case without negative effects on growth (Chapter 5.2). For both, carp and tilapia, an impact of oxalate on lipid metabolism was evident, lowering body lipid content and blood cholesterol in inclusion levels from 1.5% or higher. JKM can become a valuable alternative to present dietary protein sources in aquaculture feeds. The nutritional attributes of JKM need further research, especially longer-term testing in a commercial scenario and application in commercially produced feeds. Results of this thesis pose a useful addition to previous research and can be referred to for realizing these next steps.

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