Random amplification of polymorphic DNA (RAPD) typing of carnobacteria isolated from hindgut chamber and large intestine of Atlantic cod (Gadus morhua l.)

Autochthonous and allochthonous bacteria were isolated from hindgut chamber and large intestine of fed and starved Atlantic cod (Gadus morhua L.). All bacterial strains isolated from hindgut chamber belong to carnobacteria. However, only 10.2% of the bacteria strains from the large intestine belong to carnobacteria. Random amplification of polymorphic DNA (RAPD) analysis using three selective primers, was performed to further identify the carnobacteria strains. Nine of these were isolated from hindgut chamber contents, ten associated with epithelial cells of the hindgut chamber, and six isolated from the large intestines of fed and starved fish. The 25 isolates segregated into eight clusters. The major cluster comprised nine strains isolated from the hindgut chamber of both fed and starved fish showing low similarity with the reference strains. The other strains isolated from the hindgut were located in clusters showing high similarity with Carnobacterium gallinarum or Carnobacterium piscicola. Strains isolated from large intestine appeared more divergent and were located in five different clusters. Autochthonous (indigenous) bacteria were clearly demonstrated in the hindgut chamber as transmission electron microscopy revealed rod-shaped bacteria between adjacent microvilli. Endocytosis of bacteria by epithelial cells was observed in the hindgut chamber.     

Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity

In situ zymography is a method for the detection and localization of enzymatic activity in tissue sections. This method is used with frozen sections because routine fixation of tissue in neutral-buffered formalin inhibits enzyme activity. However, frozen sections present with poor tissue morphology, making precise localization of enzymatic activity difficult to determine. Ethanol- and zinc-buffered fixative (ZBF) are known to preserve both morphological and functional properties of the tissue well, but it has not previously been shown that these fixatives preserve enzyme activity. In the present study, we show that in situ zymography can be performed on ethanol- and ZBF-fixed paraffin-embedded tissue. Compared with snap-frozen tissue, ethanol- and ZBF-fixed tissue showed stronger signals and superior morphology, allowing for a much more precise detection of gelatinolytic activity. Gelatinolytic enzymes could also be extracted from both ethanol- and ZBF-fixed tissue. The yield, as analyzed by SDS-PAGE gelatin zymography and Western blotting, was influenced by the composition of the extraction buffer, but was generally lower than that obtained from unfixed tissue. (J Histochem Cytochem 58:29–39, 2010)     

Damaging effect of the fish pathogen <Emphasis Type="Italic">Aeromonas salmonicida</Emphasis> ssp. <Emphasis Type="Italic">salmonicida</Emphasis> on intestinal enterocytes of Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.)

In fish, bacterial pathogens can enter the host by one or more of three different routes: (a) skin, (b) gills and (c) gastrointestinal tract. Bacteria can cross the gastrointestinal lining in three different ways. In undamaged tissue, bacteria can translocate by transcellular or paracellular routes. Alternatively, bacteria can damage the intestinal lining with extracellular enzymes or toxins before entering. Using an in vitro (Ussing chamber) model, this paper describes intestinal cell damage in Atlantic salmon (Salmo salar L.) caused by the fish pathogen Aeromonas salmonicida ssp. salmonicida, the causative agent of furunculosis. The in vitro method clearly demonstrated substantial detachment of enterocytes from anterior region of the intestine (foregut) upon exposure to the pathogen. In the hindgut (posterior part of the intestine), little detachment was observed but cellular damage involved microvilli, desmosomes and tight junctions. Based on these findings, we suggest that A. salmonicida may obtain entry to the fish by seriously damaging the intestinal lining. Translocation of bacteria through the foregut (rather than the hindgut) is a more likely infection route for A. salmonicida infections in Atlantic salmon.Financial support from the Commission of the European Communities, quality of Life and Management of Living Resources programme, project Q5RT-2000-31656 Gastrointestinal Functions and Food Intake Regulation in Salmonids: Impact of Dietary vegetable Lipids (GUTINTEGRITY) and from Magnus Bergvalls Stiftelse for KS, is acknowledged.This work does not represent the opinion of the European Community, which is thus not responsible for any use of the data presented.