Flavobacterium psychrophilum est le pathogène de la maladie de l’eau froide ou du syndromede l’alevin qui provoque des pertes économiques importantes dans les élevages de truite arc-en-cielen Aquitaine. Au cours de cette thèse, nous avons développé dans un premier temps un nouveau testqPCR TaqMan en duplex qui cible les gènes (i) adnr16S de F. psychrophilum et (ii) β-actine de latruite arc-en-ciel ; il permet de déterminer le taux d’infection de tissus contaminés au cours d’unemême analyse.Dans un second temps, nous avons cherché à objectiver trois approches préventives quipourraient être mises en oeuvre pour limiter l’infection à F. psychrophilum dans les élevages. Lacaractérisation de biocides classiquement utilisés par la filière aquacole a permis de proposer auxaquaculteurs des méthodes de désinfection réellement efficaces contre F. psychrophilum mais quin’affectent pas le développement des oeufs ni des futurs alevins. En parallèle, l’impact de la mise enplace de traitement probiotiques a été caractérisé à la surface des oeufs, à la surface du mucus et/oule long du système digestif, dès les tout premiers stades de développement des truites, sur le nombreet l’intensité des crises de flavobactériose dans les élevages. Deux traitements microbiens ont étéretenus : l’E112® (DOXAL-FRANCE, Pessac) et le Bactocell® (LALLEMAND, Toulouse). Enfin, troispréparations vaccinales ont été préparées (« Adjuvant », « Autovaccin » et « Membrane Externe ») ettestées pour leur capacité à protéger les poissons de la flavobactériose. / Flavobacterium psychrophilum is the causative agent of cold water disease or the rainbow troutfry syndrome which causes important economic losses in rainbow trout breedings. During this thesis,we firstly developed a new duplex Taqman qPCR test which targets the following genes (i) F.psychrophilum adnr16S and (ii) rainbow trout β-actine. This test enables to obtain the infection rate ofcontaminated tissues, during a same analysis.Then, we tried to objectivize three preventive approaches which could be implemented inbreedings to limit the infection by F. psychrophilum. The characterization of biocides used inaquaculture allowed proposing to the fish farmers effective methods of disinfection against F.psychrophilum. These methods do not affect the development of eggs or future fish larva. Moreover,the impact of the implementation of probiotics on the surface of eggs, on the surface of the mucusand/or the digestive system, from the very first steps of trout development has been characterized onthe number and the intensity of flavobacteriosis outbreaks in the breeding farms. Two probiotictreatments were selected: l’E112® (DOXAL-FRANCE, Pessac) and Bactocell® (LALLEMAND, Toulouse).Finally, three vaccine preparations (« Adjuvant », « Auto-vaccine » and « Outer Membrane ») wereprepared and tested for their capability to protect farmed fish against flavobacteriosis.

La santé des cheptels et la maîtrise des maladies est un enjeu majeur de compétitivité des élevages et de durabilité des filières. Les maladies d'origine bactérienne sont responsables de pertes économiques importantes en pisciculture. La bactérie Flavobacterium psychrophilum, qui touche l’ensemble des salmonidés et plus particulièrement la truite arc-en-ciel (Oncorhynchus mykiss),est très largement distribuée dans le monde et en France. En l'absence de vaccin commercial efficace, la maladie est jusqu’à présent systématiquement combattue à l'aide de traitements antibiotiques. Dans un contexte d’antibiorésistance croissante, il est nécessaire de trouver d’autres moyens de lutte et la sélection d'animaux naturellement plus résistants constitue une priorité pour la filière trutticole française. Si le caractère héritable de la résistance à la maladie est bien démontré chez la truite, une meilleure connaissance de ses différentes composantes et des déterminismes génétiques sous-jacents est nécessaire pour optimiser les modalités d’introduction de la résistance dans les objectifs des schémas de sélection conduits par les entreprises françaises.Dans ce contexte, l’objectif de cette thèse est d’étudier le déterminisme génétique de différentes composantes de la réponse à l’infection par F. psychrophilum par une approche de cartographie de QTL (quantitative trait locus) ; puis d’évaluer dans quelle mesure ces déterminismes dépendent des protocoles infectieux utilisés pour tester la résistance.Pour ce faire, nous avons combiné les résultats obtenus avec des infections expérimentales par injection et balnéation et une infection naturelleEn utilisant, d’une part des croisements expérimentaux entre lignées isogéniques à la résistance contrastée et d’autre part, avec une lignée commerciale élevée dans une entreprise de sélection française.Nous mettons en évidence que la réponse de la truite arc-en-ciel à l’infection par F. psychrophilum est un caractère complexe, contrôlé par un grand nombre de QTL d’effet modéré et en interaction. Nos résultats suggèrent également que les différentes composantes de la réponse à l’infection (résistance, endurance, résilience, portage) sont en partie contrôlées par des déterminants génétiques différents, et que certains mécanismes de défense contre l’infection par F. psychrophilum dépendent de la voie d’infection (infection par balnéation ou par injection). Cette étude ouvre la voie à une meilleure compréhension des mécanismes immunitaires sous-jacents à la réponse de la truite arc-en-ciel à l’infection par F. psychrophilum, et constitue une première étape vers la mise en place de la sélection génomique pour la résistance à F. psychrophilum dans les populations de truites françaises. / Health management and disease control are of both major economic and environmental concerns for animal production systems. Bacterial diseases are responsible for important economic losses in fish farming. Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD), affects particularly rainbow trout (Oncorhynchus mykiss) and is found worldwide where salmonids are raised. As no commercial vaccine is available at the moment, the only way to fight BCWD remains through the use of antibiotic treatments. In a context of growing antibiotic resistance threat, additional methods to tackle fish disease are highly needed. Improving the natural host defense mechanisms against infectious pathogens through selective breeding is one of the priorities for French trout farmers. Moderate heritabilities were estimated for resistance against BCWD, indicating that selective breeding is a promising approach to improve trout defense mechanisms against F. psychrophilum. In order to implement disease resistance in French breeding schemes, we need to better understand the genetic determinism of this trait.In this context, the objective of this PhD is to study genetic determinism of different components of trout response to different infection protocols with F. psychrophilum using a QTL (Quantitative Trait Loci) mapping approach.To do so we used three experimental crosses between isogenic lines of trout with contrasting susceptibility. Finally, a study on a commercial line under selection in a French breeding company was performed after a natural disease outbreak to validate the interest of QTL previously detected.In this study, we showed that an important number of interacting QTL controls rainbow trout response to infection with F. psychrophilum. Our results suggest that different components of response to infection (resistance, endurance, resilience, bacterial load) might be controlled by different genetic determinisms. We also showed that different infection protocols trigger different immune mechanisms that may be specific to the route of entry of the pathogen. This study paves the way toward a better understanding of underlying immune mechanisms of rainbow trout response to F. psychrophilum. Finally, this study is the first step toward implementing genomic selection of resistance to BCWD in French rainbow trout population.

Flavobacterium psychrophilum est l’agent pathogène des flavobactérioses d’eau froide touchant essentiellement les salmonidés dont la truite arc-en-ciel Oncorhynchus mykiss d’élevage. Cette bactérie Gram négative a un très fort impact économique en aquaculture car elle peut causer jusqu'à 70 % de mortalité dans les bassins d’élevage. La flavobactériose se décline sous deux formes pathologiques : la maladie de l’eau froide touchant les poissons adultes et le syndrome de l’alevin de truite arc-en-ciel touchant les juvéniles.Au cours de se travail, une méthode de PCR quantitative a été proposée. Elle permet en moins de trois heures de détecter et de quantifier un nombre de copies du gène codant l’ARNr 16S de la bactérie dans les tissus du poisson. Cette méthode a été testée sur différentes suspensions bactériennes (F. psychrophilum, autres flavobactéries, autres pathogènes) afin d’en valider la spécificité. La sensibilité de la méthode de détection a été évaluée à 1 et 2 bactéries par PCR en fonction de la matrice biologique utilisée.Une étude écotoxicologique a été menée et montre d’une part que F. psychrophilum est une bactérie hyper-sensible au cadmium comparée aux autres bactéries Gram négatives. Sa croissance est diminuée d’un facteur 2 en présence d’une contamination au Cd à 0,4 µM. D’autre part, nous avons constaté qu’une contamination de truites juvéniles par 1 µg CdCl2/L (2 mois) et une injection de 5 × 107 flavobactéries par individu (1 mois) ne provoque aucune mortalité. L’expression génique mesurée sur ses poissons démontre que le cadmium peut avoir des effets contradictoires sur le système immunitaire du poisson, pouvant soit exacerber ou diminuer la réponse immunitaire selon l’organe considéré. Un travail comparatif de la prévalence de la flavobactérie dans 7 sites aquacoles d’Aquitaine a démontré que la flavobactérie est omniprésente et que sa pathogénicité est contrôlée par le système immunitaire des poissons en bonne santé apparente. L’expression génique mesurée sur les poissons malades et apparemment sains nous apporte deux informations importantes : 1/ les gènes codant pour la métallothionéine A et l’interleukine 1-β sont de bons bio-marqueurs de la maladie et 2/ la répression des gènes codant pour le complexe majeur d’histocompatibilité 2-β, le facteur de croissance transformant β, le cluster de différentiation 8-α et l’immunoglobuline T dans la rate des poissons malades montre un effondrement du système immunitaire acquis nous permettant d’émettre l’hypothèse que ce phénomène déclenche l’apparition de la maladie. Ainsi, F. psychrophilum aurait un comportement de pathogène à virulence latente.Afin d’imaginer de nouvelles mesures prophylactiques et pour mieux comprendre la pathogénicité de la bactérie, une analyse du protéome de la membrane externe couplée à l’annotation du génome séquencé a été effectuée. Il a été identifié entre autres 1/ des protéines d’adhésion et d’invasion des tissus et 2/ des protéines d’acquisition de métabolites de l’hôte. De plus, un nombre important de protéines immunogènes chez la truite potentiellement utilisable dans un cocktail vaccinant a été détecté. Afin de chercher un vecteur pour ce cocktail, des nanoparticules d’acide γ-glutamique et phénylalanine d’environ 100 à 200 nm de diamètre ont été synthétisées. Ces dernières constituent une approche séduisante pour vacciner les poissons avec des antigènes de F. psychrophilum encapsulés puis incorporés dans la nourriture. / Flavobacterium psychrophilum is the causative agent of cold water flavobacteriosis, a condition affecting mostly salmonid fish, including the farmed rainbow trout Oncorhynchus mykiss. This Gram negative bacterium can cause up to 70% mortality in breeding tanks and has a very strong economic impact on the fish farming industry. Flavobacteriosis can take two pathological forms: the cold water disease affecting adult fish and the rainbow trout fry syndrome affecting juveniles.In the present study, a method of quantitative PCR was devised that allowed for the detection and the quantification, within three hours, of the 16S rRNA copy number in fish tissues. This method’s specificity was confirmed through the use of various bacterial suspensions (F.psychrophilum, others flavobacteria and others pathogens) and its detection limit was estimated to be 1 and 2 bacteria in broth and in biological matrices, respectively.An ecotoxicological study was then performed that showed that, on the one hand, F. psychrophilum is cadmium hypersensitive compared to others Gram negative bacteria because its growth rate, compared to a control, is decreased by a factor 2 at a cadmium concentration of 0,4 µM. On the other hand, we observed that subjecting rainbow trout juveniles to a concentration of 1 µg CdCl2/L for2 months prior to an injection of 5 × 107 F. psychrophilum by fish didn’t lead to any mortality. The gene expression which was measured on these fish demonstrated that cadmium can have contradictory effects on the immune system of fish, which could enhance or decrease the immune response depending of the organ. A comparative work of the prevalence of flavobacteria in 7 fish farms within the Aquitaine region (France) demonstrated that the bacterium was endemic and present in asymptomatic fish. Gene expression levels were measured on diseased and asymptomatic fish and demonstrated that the genes metallothionein A and interleukine 1-β were good biomarkers of the disease and that repression of the genes major histocompatibility complex 2-β, transforming growth factor -β, cluster of differentiation α and immunoglobulin T in the spleen of diseased fish was indicative of a collapse of the acquired immune system. We therefore hypothesized that this event marked the beginning of the disease and that F. psychrophilum is mostly an opportunistic pathogen.To prepare the development of new prophylactic techniques and to understand better the bacterium pathogenicity, an analysis of the outer membrane proteome coupled with sequencing of the bacterial genome was also performed. Furthermore, a significant number of immunogenic proteins were identified as good candidates for the preparation of a vaccine. Finally, γ-glutamic acid and phenylalanine nanoparticles of about 100 - 200 nm in diameter were synthesized to serve as potential vector for this vaccine. These nanoparticles should be tested to administrate F. psychrophilum antigens to fish through the digestive route.

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

Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD), is tolerant of recommended ultraviolet (UV) doses used in recirculation aquaculture systems (RAS). Membrane filtration (MF) is used to remove pathogens from wastewater in many industries, but has not been thoroughly tested in RAS. In this study, bacterial removal efficiencies were assessed between MF and UV treatments over 30 days in an RAS. Bacterial removal efficiencies were not significantly different (p > 0.05) between MF and UV treatments, which removed 98.5 ± 0.4 % and 99.6 ± 0.1 % of total bacteria and 93.2 ± 5.2 % and 92.5 ± 4.1 % of heterotrophic bacteria, respectively. Under laboratory conditions, a MF system was challenged with concentrated doses of F. psychrophilum and achieved 5.8 ± 0.2 log reductions of the pathogen. Therefore, MF represents a potential alternative to UV irradiation and could be used to reduce the prevalence of F. psychrophilum in RAS, potentially reducing the incidence of BCWD and its impact on the aquaculture industry. / Environment Canada and Ontario Ministry of Agriculture, Food and Rural Affairs.

Examination of variation at polymorphic microsatellite loci is a widely accepted method for determining parentage and examining genetic diversity within rainbow trout (Oncorhynchus mykiss) breeding programs. Genotyping costs are considerable; therefore, we developed a single-step method of co-amplifying twelve microsatellite loci in two hexaplex reactions. The protocol is explicitly described to ensure reproducible results. I applied the protocol to samples previously analyzed at the National Center for Cool and Coldwater Aquaculture (NCCCWA) with previously reported marker sets for a comparison of results. Each marker within the multiplex system was evaluated for duplication, null alleles, physical linkage, and probability of genotyping errors. Data from four of the 12 markers were excluded from parental analysis based on these criteria. Parental assignments were compared to those of a previous study that used five independently amplified microsatellites. Percentages of progeny assigned to parents were higher using the subset of eight markers from the multiplex system than with five markers used in the previous study (98% vs. 92%). Through multiplexing, use of additional markers improved parental allocation while also improving efficiency by reducing the number of PCR reactions and genotyping runs required. I evaluated the methods further through estimation of F-statistics, pairwise genetic distances, and cluster analysis among brood-years at the NCCCWA facility. These estimates were compared to those from nine independently amplified microsatellites used in a previous study. Fst metrics calculated between brood-years showed similar values of genetic differentiation using both marker sets. Estimates of individual pairwise genetic distances were used for constructing neighbor-joining trees. Both marker-sets yielded trees that showed similar subpopulation structuring and agreed with results from a model-based cluster analysis and available pedigree information. These approaches for detecting population substructure and admixture portions within individuals are particularly useful for new breeding programs where the founders' relatedness is unknown. The 2005 NCCCWA brood-year (75 full-sib families) was challenged with Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD). The overall mortality rate was 70%, with large variation among families. Resistance to the disease was assessed by monitoring post-challenge days-to-death. Phenotypic variation and additive genetic variation were estimated using mixed models of survival analysis. The microsatellite markers used were previously isolated from BAC clones that harbor genes of interest and mapped onto the rainbow trout genetic linkage map. A general relationship between UBA gene sequence types and MH-IA-linked microsatellite alleles indicated that microsatellites mapped near or within specific major histocompatibility (MH) loci reliably mark sequence variation at MH genes. The parents and grandparents of the 2005 brood-year families were genotyped with markers linked to the four MH genomic regions (MH-IA, MH-IB, TAP1, and MH-II) to assess linkage disequilibrium (LD) between those genomic regions and resistance to BCWD. Family analysis suggested that MH-IB and MH-II markers are linked to BCWD survivability. Tests for disease association at the population level substantiated the involvement of MH-IB with disease resistance. The impact of MH sequence variation on selective breeding for disease resistance is discussed in the context of aquaculture production. / Master of Science