Universiẗat, Diss., 2002--Mainz.

Orientador: Manoel Victor Franco Lemos / Banca: Maria Inês Tiraboshi Ferro / Banca: Maria José Tavares Ranzani de Paiva / Resumo: 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 / Abstract: 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 / Mestre

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.

Die vorliegende Dissertation befasst sich mit den Struktur-Eigenschafts-Beziehungen von sternförmigen Mesogenen mit kontrollierbaren Konformationen in den LC-Phasen. Zunächst sollte mithilfe verschiedener Moleküldesigns geklärt werden, wie eine Faltung der Arme verhindert werden kann, und somit, ob sternförmige Konformationen in den kolumnaren Packungen realisiert werden können. Hierzu wurde erfolgreich eine Bibliothek von dreiarmigen Amidsternen, semiflexiblen Oligoestersternen mit hexasubstituiertem Benzolkern und formtreuen hexasubstituierten Benzolen synthetisiert. Die besondere Herausforderung bei der Darstellung letzterer lag in der C3-Symmetrie der Verbindungen und konnte durch Optimierung der Synthesestrategie mittels aufeinander folgender Wittig-Horner- und Suzuki-Reaktionen in einem divergenten Ansatz gemeistert werden. Ein herausragendes Ergebnis ist die Flüssigkristallinität dieser formtreuen hexasubstituierten Strukturen, wenn sie mindestens neun bzw. zwölf periphere Ketten besitzen. Die detaillierte Auswertung der Kolumnendurchmesser mithilfe von äquatorialen Reflexen sowie der Dichte und der meridionalen Beugungsmuster zeigen, dass lediglich für die formtreuen hexasubstituierten Benzolderivate eine Faltung verhindert werden kann. Intrinsische Freiräume (Kävitäten) zwischen den Oligo(phenylenvinylen)-Armen werden durch außergewöhnliche Dimerenbildung und helikale Packung der Moleküle kompensiert. In die Kavitäten der Trispyridylverbindungen können Carbonsäure-funktionalisierte Gäste unter Ausbildung von Wasserstoffbrücken eingelagert werden. Mit zunehmender Gastkonzentration wird die helikale Dimerphase des Wirts kontinuierlich in eine neue kolumnare Phase von monomeren Supermesogenen ohne helikale Struktur umgewandelt. Da die Gäste in den Supermesogenen vollständig von den Oligo(phenylenvinylen)-Armen und den aliphatischen Ketten umschlossen sind, handelt es sich bei der Wirtverbindung erstmals um einen flüssigkristallinen Endorezeptor mit drei Bindungsstellen. Das Sternmesogen mit größeren intrinsischen Freiräumen ermöglicht die Einlagerung von funktionalen Bausteinen wie z.B. Anthracenchromophoren. Aus Untersuchungen mittels Festkörper-NMR- und Fluoreszenzspektroskopie geht hervor, dass sich die Mesophase mit drei Anthracengästen langsam in eine doppelt nanosegregierte Struktur umwandelt, in der intrakolumnar Oligo(phenylenvinylen)-Arme und Anthracene Seite an Seite segregiert stapeln und so segmentierte Kolumnen bilden. Diese Art von doppelter Nanosegregation offenbart das Potential des verwendeten Moleküldesigns im Bezug auf die Entwicklung mesomorpher Multikabelstrukturen. Im Vergleich zu den Supermesogenen weisen die analogen Sternverbindungen mit kovalent gebundenen Pseudogästen um über 100 °C höhere Klärpunkte auf, was unter Berücksichtigung der strukturellen Ähnlichkeit der kolumnaren Phasen und der ähnlichen Mischungsenthalpien in unterschiedlichen Werten der Mischungsentropie begründet liegen muss. Der Vergleich mit einer 1:3-Mischung ohne spezifische Wirt-Gast-Wechselwirkung bestätigt in diesem Zusammenhang den Einfluss der Bindungsart der Gäste auf die Mesophasenstabilität. Die Klärtemperaturen der Sternmesogene lassen sich folglich über die Art der Bindung der Gastmoleküle kontrollieren. Dies ist vor allem für die Orientierung kolumnarer Phasen in dünnen Filmen großer funktionaler Mesogene, die häufig erst bei sehr hohen Temperaturen unter Zersetzung in die isotrope Phase übergehen, interessant. / This dissertation examines the structure-property relationships of star-shaped mesogens with controllable conformations in the LC phases. First it should be clarified by means of different molecular designs, how a folding of the arms can be prevented, and thus, if reliable star-shaped conformations can be realized in the columnar stacks. For this reason, a library of three-armed amide stars, semi-flexible oligoester stars with a hexasubstituted benzene core and shape-persistent hexasubstituted benzenes was successfully synthesized. The particular challenge in the preparation of the latter was the C3-symmetry of the compounds and could be accomplished by optimizing the synthetic strategy using subsequently Wittig-Horner and Suzuki reactions in a divergent route. An outstanding result is the liquid crystallinity of these shape-persistent hexasubstituted structures, if they contain at least nine or twelve peripheral chains. The detailed evaluation of the column diameters, using equatorial reflections, as well as the density and the meridional scattering patterns shows that only the shape-persistent hexasubstituted benzene derivatives can be prevented from folding. Intrinsic free spaces (cavities) between the oligo(phenylenevinylene) arms are compensated by exceptional dimer formation and helical packing of the molecules. Carboxylic acid functionalized guests can be incorporated into the cavities of the trispyridyl compounds by forming hydrogen bonds. Upon increasing the guest concentration the helical dimer phase of the host is continuously converted into a new columnar phase of monomeric supermesogens without a helical structure. Since the guests of the supermesogens are entirely enclosed by the oligo(phenylenevinylene) arms and the aliphatic chains, the host compound is the first liquid crystalline endoreceptor with three binding sites. The star mesogen with larger intrinsic free spaces facilitates the incorporation of functional building blocks such as anthracene chromophores. Investigations by solid state NMR and fluorescence spectroscopy uncovered that the mesophase with three anthracene guests slowly converts into a double nanosegregated structure in which oligo(phenylenevinylene) arms und anthracenes stack side by side and thus form segmented columns. This type of double nanosegregation reveals the potential of the molecular design used with respect to the development of mesomorphic multi cable structures. In comparison to the supermesogens the analog star compounds with covalently bound pseudo guests exhibit more than 100 °C higher clearing points. Considering the structural similarity and the similar mixing enthalpy this must be due to different values of the mixing entropy. In this context, the comparison with a 1:3 mixture without any specific host-guest interaction confirms the influence of the binding mode of the guests on the mesophase stability. As a consequence, the clearing temperatures of the star mesogens can be controlled by the binding mode of the guest molecules. This is particularly interesting for the orientation of columnar phases in thin films of large functional mesogens which frequently transform into the isotropic phase at very high temperatures under decomposition.

Flavobacterium columnare yra gerai žinomas patogenas sukeliantis kolumnariozę, ir kurio sukeliama liga paliečia gėlavandenes žuvis: tiek akvariumines, tiek žuvivaisos įmonėse, tiek gyvenančias natūraliose gamtinėse sąlygose. Bakteriją pirmą kartą 1922 metais aprašė Herber Spencer Davis, ir jai suteikė pavadinimą, kuris vėliau kelis kartus keitėsi (Bacillus columnaris, Cytophaga columnaris, Chondrococcus columnaris, Flexibacter columnaris) kol buvo pripažintas Flavobacterium columnare pavadinimas, kuris dabar naudojamas. Jungtinėse Amerikos Valstijose ši bakterija atneša labai didžiulius nuostolius. Registruotais duomenimis 2003 m. kolumnaris liga nusinešė daugiau nei 30 milijonų JAV dolerių vien tik iš šamų pramonės. F. columnare dažniausiai pasižymi tuo, kad sukelia žiaunų nekrozę, pelekų erozijas ir žvynų pažeidimus. Išauginta ant Cytophaga agaro kolonija dažniausiai būna rizoidinio tipo, adhezyvi, gelsvos spalvos (dėl pigmento fleksirubinino). Raudonųjų Kongo dažų absorbcija – vienas labiausiai patikimiausių biocheminių testų. Taip pat egzistuoja ir kiti diagnozavimo metodai – serologiniai, molekuliniai. Šios ligos prevencijai reikia reguliariai naudoti kalio permanganatą, vario sulfatą ar kitą dezinfekcinę medžiagą, kuri naikina F. columnare bakterijas. Jei tenka gydyti – visai neseniai pasirodė tyrimai, kuriuose buvo aprašytas fagų panaudojimas. Jie pasižymi kolonijų augimą stabdančiu ar bakterijas naikinančiu poveikiu, tačiau dar reikia atlikti daugybę tyrimų norint... [toliau žr. visą tekstą] / Flavobacterium columnare is a well known worldwide pathogen, causative agent of Columnaris disease, elicting significant problems in freshwater fish including aquariums, culture places, natural surroundings. The first time the bacteria was described and named by Herber Spencer Davis. The name has changed for several times till reached the one is used now (Bacillus columnaris, Cytophaga columnaris, Chondrococcus columnaris, Flexibacter columnaris). In the industry of USA catfish this pathogen causes millions of dollars of losses. According to the statistics of the year 2003, the industry lost more than 30 millions of dollars of income. F. columnare in fish infections may cause skin lesions, fin erosions and gill necrosis, with a high degree of mortality. Cultured on Cytophaga agar grows mostly in rhizoit type very much adhesive collonies coloured in yellow due to pigment flexirubinin. One of the most revieling biochemical tests – absorbance of Congo red dye. Also it can be diagnosed by many serological and molecular assays. Preventive control should be used regularly involving potassium permanganate, copper sulphate or other desinfecant, which is available for F. columnare on the market. The latest treatment described for columnariosis is based upon phages which prevent the growth of F. columnare or kill it, but there are many studies to be done before making any conlusions. The aim of the research – to evaluate morfophysiological status and infestation with Flavobacterium... [to full text]

Techn. Universiẗat, Diss., 2000--Berlin.

A Catfish Management Database (CMD) was developed to analyze data from large commercial catfish farms. The CMD was developed so that data collected by the farm could be used for management of the farm and for identifying some of the risk factors associated with important bacteria diseases. This database was designed to 1) to incorporate production data already being recorded for generating reports for use at weekly managerial meetings focused on feeding rates, feed conversion ratios, mortalities and harvesting events 2) be easily used by a catfish farmer to collect management data in order to analyze production efficiency through a series of farmer defined management reports and 3) provide the farm with easy access to management reports. Additional customized reports can be generated as requested by the farm management. The next objective of this research was to determine pond level risk factors associated with columnaris disease and Enteric Septicemia of Catfish related mortalities. The data from the CMD was used to produce two publications detailing the analysis of the data and production of a univariate and multivariate models of pond level risk factors associated with both diseases. These studies showed some commonly recorded production variables were associated with either columnaris and/or ESC associated mortalities and if monitored could help identify “at risk” ponds prior to disease outbreaks. A study was then conducted to examine the cost associated with mortality on Mississippi commercial catfish farms. The mortalities examined included ponds that had mortalities from columnaris disease, ESC and then any ponds that had mortalities from either. The cost of each disease was determined along with other factors such as pond age, feed conversion ratio and feed cost that influence the profitability of a commercial catfish farm.

The 16S, 23S, and 5S ribosomal RNA (rRNA) genes are highly conserved sequences in bacteria. For this reason, rRNA genes are often used for phylogenetic classification. On the other hand, the regions between the structural sequences, known as intergenic spacer regions (ITS), are under less evolutionary pressure to be conserved. Because they are not as highly conserved, they can be used to differentiate strains of the same bacterial specie. The purpose of this study was to evaluate the 16S-23S ITS of Flavobacterium columnare, an important pathogen of cultured fish, by comparing the 16S-23S ITS sequences from 70 isolates. We developed two PCR assays that amplify overlapping regions of one large previously identified ITS. The primers targeted the 16S sequence and isoleucine tRNA encoding sequences and the 23S sequence and alanine tRNA encoding sequences. The PCR products were cloned and sequenced. We also targeted I-CeuI restriction fragments from the ATCC type strain that were separated by pulse field gel electrophoresis and analyzed the 16S-23S ITS regions. We found that the genome of this species harbors at least 6 intergenic spacer regions that are very similar and contain the same tRNA encoding sequences. This suggests that earlier studies that used the ITS for distinguishing between strains of Flavobacterium columnare may be comparing sequences from different structural RNA operons and thus have misleading data.

The catfish industry suffers losses primarily due to enteric septicemia of catfish and columnaris disease caused by Edwardsiella ictaluri and Flavobacterium columnare, respectively. Understanding the host-pathogen interactions is vital for prevention and eradication of these diseases. Hence, the overall objective of this study was to analyze whole cell proteomes of these two bacteria, and to determine the changes in E. ictaluri protein expression against in vitro iron-restriction and host serum treatment. High-throughput proteomic analysis of these bacteria was conducted using two-dimensional liquid chromatography followed by electrospray ionization tandem mass spectrometry (2-D LC ESI MS/MS) and two-dimentional gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-oflight mass spectrometry (2-DE MALDI TOF/TOF). Identified proteins were clustered into functional groups using clusters of orthologous groups, and subcellular locations as well as possible functional relationships were determined. A total of 788 unique E. ictaluri and 621 unique F. columnare proteins were identified, which represented 12 and 28 pathways, respectively. Vertebrate hosts tend to chelate free iron of their body and make the environment hostile for bacteria. Hence, reduced availability of iron may cause significant stress for pathogens and is considered a signal that leads to alteration in virulent gene expression. Similarly, E. ictaluri might use the catfish blood stream effectively for quick systemic invasion. Hence, exposure to catfish serum components might reveal the ability of E. ictaluri to protect against host defense mechanisms. Using two-dimensional difference gel electrophoresis, responses of E. ictaluri due to in vitro iron-restriction and host serum treatment were determined. A total of 50 and 19 proteins were identified to be differentially expressed due to in vitro iron-restriction and catfish serum treatment, respectively. Among the differentially expressed proteins, several putative virulent determinants, immunogenic proteins, chaperones, and housekeeping genes were noted. To initiate functional studies, four differentially expressed E. ictaluri genes (lamB, glyS, malE, and sdhA) were mutated by inrame deletion. Results from this study provided experimental evidence for many predicted proteins. In addition, identification of differentially expressed proteins provided targets for further functional analysis, which could help elucidate pathogenic mechanisms of E. ictaluri.