Thesis (Doctoral)--Universität Hannover, 1983.

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.

Flavobacterium columnare is a Gram negative fish pathogen that causes columnaris disease, which infects populations of wild and cultured fish species. However, pathogenic mechanisms of F. columnare are largely unknown. The purpose of this research is to obtain the complete sequence of the F. columnare ATCC 49512 genome to advance pathogenesis research and increase our understanding of this pathogen. To accomplish this, genome sequencing by using Sanger and 454 sequencing was conducted. The sequences were assembled, gaps were filled, and the circular genome was autoannotated. The F. columnare genome size is 3.2 Mb and AT rich (68.5% AT). It contains 2,882 predicted proteins, 71 tRNA genes and five ribosomal RNA operons. More than half (57.1%) of the open reading frames have assigned function, which included chondroitin AC lyase, proteases, collagenases, and genes involved in biofilm formation, secretion systems, iron acquisition, and gliding motility.

Aquaculture has potential to support the food supply of increasing world population. Flavobacterial diseases pose a serious problem in wild and aquacultured fish stocks throughout the world. Flavobacterium columnare, F. branchiophilum, and F. psychrophilum are well-known Flavobacterium species that cause important fish losses. Recently, new Flavobacterium species, isolated from diseased fish, have been reported, but their virulence mechanisms are not clear. Thus, the goal of this study was to understand pathogenicity of Flavobacterium species. To this goal, 86 Flavobacterium genomes were analyzed by comparative genomics. Predicted virulence genes were identified for all genomes. For each species, unique and shared virulence genes were determined. For all genomes, unique and common predicted antibiotic resistance genes were identified as well. Secreted proteins are important virulence factors. Thus, all encoded secretion and related systems were determined. By using different genomics approaches, F. columnare genomovar I (highly virulent to cold-water fish species like trout) and genomovar II (extremely virulent to warm-water fish species such as catfish and tilapia) genomes were analyzed, and transposon mutants using Tn4351 in six F. columnare genomovar II strain 94-081 were generated. The hemolysin and glycine cleavage protein mutants had 15% and 10% mortalities, respectively while wild-type strain caused 100% mortality. Potential virulence genes, unique proteins, and other genomic features of F. columnare genomovars were determined. Mutants targeting unique genes in valine-leucine-isoleucine biosynthesis pathway were constructed. The virulence of Fcol(DELTA)leuD and Fcol(DELTA)ilvD mutants exhibited reduced virulence.

Flavobacterium psychrophilum (Fp), the aetiological agent of rainbow trout fry syndrome (RTFS) and bacterial cold-water disease (BCWD), is responsible for significant mortalities and economic losses in the salmonid aquaculture industry worldwide. Currently, there is no effective commercial vaccine against RTFS available, and the treatment of the disease depends on the oral administration of a wide range of anti-microbial compounds, some of which have proven ineffective. With unsuccessful disinfection procedures, possibilities of antibiotic resistance developing and no commercial vaccine available, there is an increased need to rapidly detect Fp and reduce mortalities in the industry by improving control measures in the farm system. The aim of this thesis was to investigate possible sources of Fp in a rainbow trout fry farm system and to use this data to develop strategies to reduce the prevalence of the pathogen with this farming system. Novel assays to detect Fp (loop-mediated isothermal amplification; LAMP), quantify Fp (quantitative PCR; qPCR) and to detect the fishes’ host response to Fp (Luminex™) were developed, and then used alongside bacterial culture and nested PCR to determine the prevalence of Fp on a commercial fish farm. Four batches of eggs from 3 different geographic sources were collected on arrival at the farm and tested for the prevalence of Fp. Fry from these batches were monitored as they grew and were moved to different sites at the farm. Kidney, spleen and blood were collected at 3 different life stages from the fry, until they were sold for ongrowing by the farm. Water samples from the inlet, outlet and fry tanks were collected at each sampling point. PCR analysis and bacteriology were the two main methods selected for screening the eggs and fry tissue for Fp. All sources of eggs were found to be positive for Fp with prevalences ranging from 1.1 % - 1.9 % and there was a significant increase in prevalence over time for all 4 batches of eggs ranging from 19.8 % - 34.6 % by the final life stage sampled. There was also a substantial difference in the numbers of fry samples positive for Fp depending on whether nested PCR or bacterial culture were used, as well as the organ (kidney or spleen) tested. This highlighted the importance of sampling both organs rather than just the one. Nested PCR was more sensitive than culture with 13 % of the fry samples reported as Fp positive, by sampling both the kidney and spleen collectively, while only 5 % were Fp positive by bacteriology. The levels of Fp in all samples could not be quantified by qPCR due to limits in the sensitivity of the assay. For those samples that were quantified at the levels of Fp detected by qPCR ranged from 3.38 x 104 well-1 - 2.07 x 106 well-1 genome copies in egg samples; from 3.38 x 103 well-1 – 3.07 x 107 well-1 genome copies well-1 in tissue samples (spleen or kidney), and from 7.89 x 103 – 7.22 x 104 genome copies well-1 in water samples. The sensitivity of the standard curve was limited to 103 copies well-1 and following optimisation of the assay the annealing temperature was decreased by 1˚C to 62°C to reduce the cross-reactivity to negligible levels, though this reduced the sensitivity of the assay even further to 104 copies well-1. The detection limits by qPCR obtained by spiking samples with known amounts of Fp were 192 CFU mg-1 from egg samples, 184 CFU mg-1 from fry tissue samples, and 220 CFU ml-1 from water samples,. The sensitivity of the LAMP assay determined by spiking egg, kidney, spleen and water samples was 18 CFU mg-1, 22 CFU mg-1, 25 CFU mg-1 and 16 CFU ml-1, respectively. The latter was similar to, though not as sensitive as nested PCR. Nested PCR limits determined by spiking egg, kidney, spleen and water samples were 14 CFU mg-1, 11 CFU mg-1, 13 CFU mg-1 and 11 CFU ml-1. No cross-reactivity was found with any bacteria including other Flavobacterium species with nested PCR but cross-reactivity with other Flavobacterium species were found with both qPCR (1.51 % with Flavobacterium aquatile and 0.30 % with Flavobacterium johnsoniae) and LAMP. The LAMP assay showed slight cross-reactivity with Flavobacterium columnare and Flavobacterium branchiophilum. A novel Luminex™ assay was also developed and optimised, using microspheres coated with Fp, to detect antibodies to Fp in the serum of the fry. The Luminex™ allowed small volumes of serum from individual fry to be used to evaluate antibody response as an indirect method to determine exposure to and infection by Fp. A large number of fry from all 4 batches (88% - 94%) of eggs were found to contain anti-Fp antibodies though it still remains to be determined whether the antibodies were specific to Fp. From the work carried out in this study, it can be concluded that whether eggs are carrying Fp inside and/or on their surface, it should be possible to reduce the prevalence of Fp in farm systems by regularly screening the broodstock, eggs and fry. Supply of Fp-free eggs and milt is essential to reduce the reservoir of Fp on farms. Both the qPCR and LAMP assay require further optimisation but they do offer potential for the future screening of Fp at farm sites and in the laboratory. Future control measures for RFTS should include the screening of broodstock and eggs by sensitive methods so that Fp-free seed can be supplied to farms. This, alongside effective disinfection procedures, rigorous husbandry practices and future vaccine development will all be required to manage this very significant fish disease.

Thesis (Ph.D.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references (ℓ. 104-128)

Flavobacterium columnare, a Gram-negative rod-shaped bacterium, is the causative agent of columnaris disease in a variety of fish hosts but is of particular significance to the catfish industry located in the southeastern United States. Columnaris infections are a leading cause of mortalities in catfish ponds, occurring alone or in conjunction with other diseases. Typical diagnostic methods for columnaris infections involve the use of selective media following the observation of gross signs of disease. A real-time quantitative PCR (qPCR) assay to estimate the quantity of bacteria present in environmental and tissue samples was developed and validated. The genetic variability seen in F. columnare makes detection of isolates from different genomovars (genetic groups) essential to an assay for diagnostic application. Isolates from catfish generally fall into one of two different genomovars, one being virulent to catfish, while the other genomovar is thought to be largely opportunistic. The qPCR assay described herein was designed specifically to detect F. columnare isolates from the two major genomovars most often associated with farm-raised catfish. The assay was shown specific to F. columnare, regardless of genomovar, and demonstrated sensitivity consistent with similar qPCR assays. In addition, the assay provides quantitative information, estimating the bacterial loads in fish tissue and the environment. Two different applications of the assay are presented: (1) Estimate bacterial burden in fish tissue following immersion challenges to identify variation in transmission rates between channel and blue x channel hybrid catfish, and (2) Estimate the environmental burden of F. columnare in catfish ponds over the course of a single calendar year. This assay will provide an invaluable tool for researchers and diagnosticians in expanding our understanding of F. columnare and how it interacts with the host and environment.

Flavobacterium columnare is a freshwater fish bacterium responsible for columnaris disease, the second leading cause of mortality in pond raised catfish in the southeastern United States. Pulsedield gel electrophoresis (PFGE) is a particularly powerful tool in epidemiology and is now regarded as the gold standard for molecular typing of microorganisms. We developed methods for conducting PFGE on F. columnare, and determined its efficacy for characterizing F. columnare strains isolated from different locations in the Southeastern United States. Virulence diversity was observed in two different immersion challenge experiments conducted with 16 different isolates in channel catfish fingerlings. A direct correlation was found between the PFGE clustered groups and virulence. In summary, our results suggest that two genetic divisions of F. columnare channel catfish isolates exist, one that contains strains that are “primary” pathogens of channel catfish (Group A), and another that are “secondary” or opportunistic pathogens of catfish (Group B).

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-22Bitstream added on 2014-06-13T20:35:47Z : No. of bitstreams: 1 sebastiao_fa_me_jabo.pdf: 674003 bytes, checksum: 55712af3de8f36c8c3d3b36dce7dcbc0 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Dentre as enfermidades de importância na piscicultura, destaca-se a columnariose, cujo agente etiológico é a Flavobacterium columnare, bactéria de ampla distribuição geográfica, responsável por um elevado número de mortalidade em peixes de várias espécies, principalmente em condições intensivas de criação. Visando o melhor conhecimento desta bactéria para desenvolvimento de métodos de diagnóstico e controle da doença, os objetivos deste estudo foram isolar, caracterizar bioquímica e molecularmente por PCR-RFLP do gene 16S rDNA de F. columnare, detectar fenotipicamente a formação de cápsulas destes isolados pelo teste Agar vermelho congo, e avaliar a composição do EPS quando produzidos por meio de cromatografia líquida de alta eficiência. Ao todo foram obtidos 37 isolados e a caracterização bioquímica indica que os isolamentos são classificados como F. columnare. O filograma gerado pela técnica de PCR-RFLP mostrou três principais ramificações entre os isolados de F. columnare. Os testes comprovaram que a presença de cápsula na célula bacteriana não está diretamente relacionada à formação de biofilme, e o monossacarídeo preponderante em F. columnare é a glicose.Portanto, a utilização da PCR-RFLP para a identificação da bactéria apresentou-se como ferramenta mais rápida que as técnicas bioquímicas atuais e os dados referentes a produção de biofilme são relevantes para futuros estudos que busquem métodos enzimáticos para impedimento da aderência e formação de biofilmes destes patógenos aquáticos em sistemas de aqüicultura e consequentemente a prevenção da columnariose / Columnaris disease stands out among the illnesses of importance in fish breeding, its etiological agent is Flavobacterium columnare, which has been recognized as a worldwide pathogen, responsible for high degree of mortality in many fish species, especially in conditions of intensive breed. Looking for a better knowledge of this bacteria and aiming to develop diagnosis methods and disease control, the objectives of this study were to isolate, to biochemistry and molecularly characterize by 16S rDNA gene PCR-RFLP of F. columnare, to detect phenotipically the formation of capsules by the agar Congo red method, and to evaluate the EPS composition by high-performance liquid chromatography. There were obtained 37 isolates and the biochemistry characterization indicated that the isolates were classified as F. columnare. The phylogenetic tree generated by PCR-RFLP technique showed three main branches among the F. columnare isolates. The presence of capsule on the bacterial cells has not a direct relationship to biofilm formation, and considering its composition it was observed that the preponderant monosaccharide is glucose. Therefore, the PCR-RFLP alternative to identify this bacteria presented itself as a faster tool than actual biochemical techniques and the results regarding to biofilm production are relevant to future studies that search for enzymatic methods to abolish the adherence and biofilm formation by this aquatic pathogen in aquaculture systems, and, consequently, columnaris disease prevention

Walleye (Sander vitreus M.) are valuable in commercial and recreational fisheries and are affected by bacterial, fungal and viral disease. Pattern recognition receptors (PRRs) are germline-encoded and constitutively expressed and bind non-self or altered-self for immune recognition. Walleye were hypothesised to have circulating PRRs that were capable of binding diverse pathogens. These PRRs were hypothesised to increase with infection, be distributed in immunologically relevant tissues and to be strain and age specific. PRR binding was measured by affinity chromatography, plasma binding assays,SDS-PAGE, Western blots, ELISA, PCR, and immunohistochemistry. ELISA and affinity chromatography assays were developed in rainbow trout (Oncorhynchus mykiss) with known PRRs. Trout ladderlectin was confirmed as a PRR binding viral hemorrhagic septicemia virus (VHSV). These techniques were adapted to walleye using Flavobacterium columnare, chitin, VHSV and Sepharose resin. A 22 kDa protein bound to F. columnare, a 17 kDa protein bound to chitin and a 34 kDa protein bound to VHSV were identified as similar to bass apolipoprotein, carp C3 and rainbow trout intelectin, respectively. PCR and 3'-RACE-PCR were used to generate nucleotide sequence to confirm identity of walleye apolipoprotein and mannose-binding lectin (MBL)-like protein from the intelectin-like sequence. Two rabbit polyclonal antibodies were raised to 34 and 67 kDa MBL amino acid sequences and used to verify MBL-like protein as a PRR for VHSV. Healthy walleye MBL-like protein plasma concentration was 7.5 ng/ml. Significant differences were found between geographically distant strains of walleye. An ELISA demonstrated that MBL-like protein had significant differences in binding affinity between multiple strains of VHSV and different viruses found in Ontario. MBL-like protein plasma levels increased with initial infection of naïve fish with waterborne and IP VHSV (107 pfu) but did not change with IP reinfection. Previous infection with VHSV significantly decreased walleye mortality. IHC of walleye shows MBL-like protein is distributed in epithelial surfaces, primarily skin, oropharynx, gill, gastrointestinal system, renal nephrons, connective tissue of gonads and plasma. There was no qualitative difference in MBL-like protein tissue distribution in healthy and VHSV-infected walleye. This is the first evidence for fish lectins binding viruses.