Isolation And Identification Of The Pathogenic Bacteria From Diseased Turbot, And The Histopathology And Immunohistochemistry Of The Turbot

The introduction of turbot from foreign is a success. It ensures fast growth, high disease resistance and low feed coefficiency, it is of high economic value, it is well-liked with the aquatic breeding farmers. But the accommodation of fish and some shortages in culture result in some diseases, most of which are bacterial diseases.Three strains were isolated from diseased turbot during the first quarter of the year of 2007. They are Aeromonas salmonicida subsp. Achromogenes (designated as db14), Vibrio ichthyoenteri(designated as da3)and Shewanella putrefaciens.(designated as db6)respectively.Strain db14 was isolated from liver of diseased turbot. It is Gram-negative, rod shaped. Pathogenicity assays revealed that db14 was virulent to turbot and zebrafish (Danio rerio) by intraperitoneal injection challenge, with the LD50 being calculated as 1×105 cfu/fish(25g)and 1.3×102 cfu/fish. The 16S rRNA gene sequence of strain db14 demonstrated high similarity with Aeromonas salmonicida subsp. Achromogenes. Phylogenetic analysis showed a clear association of strain db14 with Aeromonas salmonicida subsp. Achromogenes. Additional evidence of the identification included in morphological, physiological and biochemical data. This strain was susceptible to fortum, rocephin amicacin, chloramphenicol and resistant to a lot of antibiotics. The tissue of infected fish demonstrated significant pathology in liver, kidney and intestine. Tissue edema and protein-like deposition can be observed in liver. The histiocytes were necrotic and the renal tubules were dropsical. The lamina propria of the intestine shrinked.Strain da3 was isolated from cholecyst of diseased turbot. It is Gram-negative, rod shaped. Pathogenicity assays revealed that da3 was virulent to turbot by intraperitoneal injection challenge. The 16S rDNA gene sequence of strain da3 demonstrated high similarity with Vibrio ichthyoenteri. Phylogenetic analysis showed a clear association of strain da3 with Vibrio ichthyoenteri. Additional evidence of the identification included in morphological, physiological and biochemical data. The tissue of infected fish had significant pathology in liver, kidney spleen, brain and intestine。In the liver, the cells deformed, deeply stained and. In the kidney, the cells was basophilic, the glomeruluses and renal tubules atrophy. In the intestine, the beaker cell swelled, the propria shrinked. There was vacuolization in the tissue of brain. There were erythropenia in liver, kidney and spleen.Strain db6 was isolated from kidney of diseased turbot. It is Gram-negative, rod shaped. Pathogenicity assays revealed that db6 was virulent to zebrafish (Danio rerio) by intraperitoneal injection challenge. The 16S rDNA gene sequence of strain db6 demonstrated high similarity with Shewanella putrefaciens. Phylogenetic analysis showed a clear association of strain db6 with Shewanella putrefaciens. Additional evidence of the identification included in morphological, physiological and biochemical data. Shewanella putrefaciens is a kind of aquatic bacteria, but it is more usually a pathogen of human than fish. This strain was susceptible to fluperacid. The tissue of infected fish demonstrated significant pathology in liver, kidney and intestine.Each of the above 3 strains was firstly reported as a pathogen of turbot. It reflected an important problem to be solved that the turbot is faced with more and more pathogens.Regarding the localization of the bacteria in the tissue of the fish, it is seldom studied in our country. We located the pathogen in the turbot infected by Edwardsiella tarda using an indirect immunofluorescent and Gram's stain technology.We found the liver, kidney and spleen of diseased turbot changed significantly, and there were only few bacteria in the organs. There were many bacteria in the connective tissue covering the organs. The distributions of bacteria showed different characteristics in different sites: In the interior of the organ, it existed in the macrophage as a colony, in the intracellular substance of the connective tissue covering the organs, the bacteria can exist by single, in pairs or form a colony. The bacteria may damage the cells by secreting exotoxin, for the bacteria were not so many in the damaged tissue.