Aquaculture Europe 14, San Sebastian, Spain, 14 - 17 October 2014, pp.36-37
Turkey is one of the leading countries in the aquaculture of European sea bass (Dicentrarchus labrax) but mainly bacterial diseases occurring in fish farms limits the amount and development of this production. Vibriosis, which is caused mainly by the fish pathogen V. anguillarum is among the most fatal diseases. Characterization of this species is important for the development of more effective protection strategies. The aim of this study is the characterization and determination of the biofilm formation of Vibrio anguillarum isolates that induced vibriosis in cultured European sea bass reared in marine farms operating in Aegean Sea region.
Material and Methods
In this study, sampling studies were made on 8 fish samples weighing between 150-200 g, showing some disease signs supplied from two farms. After anahestesia, fish samples were dissected under aseptic conditions and bacteriological inoculations from the internal organs of fish samples were streaked onto TSA (%2 NaCl) and incubated for 4 days. Bacterial isolates were identified by using morphological and biochemical properties and API 20E strips were also used additionally. Molecular confirmation of the identification was made by using rpoN-PCR method which is a species-specific method and performed by using rpoN-ang5’ ve rpoN-ang3’ primers (Gonzalez et al., 2003). Serotype of the V. anguillarum isolates were determined by using slide agglutination test (Sorensen and Larsen, 1986) and dot-blot test (Cipriano et al., 1985) in which the antibodies raised against V. anguillarum serotypes O1, O2 and O3 were used. Biofilm formation ability of V. anguillarum isolates on ELISA plate and pellicle formation in liquid-air interface were also investigated (Woodward et al., 2000).
Diffuse hemorrhages on the body surface and especially in the anal region, loss of scales, exofthalmia, hemorrhages in the gills and ascites in the abdomen were observed in the external examination of the diseased fish samples. Accumulation of a fluid in the abdominal cavity, diffuse hemorrhages and hyperemia over the liver, thinning and transparency on the hyperemic intestine wall and enlarged spleen were observed in the internal examination of the diseased fish samples. After bacteriological sampling studies, V. anguillarum was recovered from the fish samples obtained from both fish farms visited in this study. V. splendidus I, V. harveyi, V. orientalis, V. mediterranei, V. alginolyticus and Aeromonas schubertii were recovered besides V. anguillarum isolates from farm (A) and A. schubertii was recovered besides V. anguillarum isolates from farm (B) as secondary bacterial agents. Isolates that were identified as V. anguillarum formed a single band of 519 bp in the rpoN PCR analysis and subsequently molecular confirmation of the biochemical identification was made. In the slide agglutination test, O-antigens extracted from V. anguillarum recovered from diseased cultured sea bass in this study, only reacted with the antibody raised against V. anguillarum serotype O1 and produced agglutination. As the reference V. anguillarum isolates and field isolates has reacted with the polyclonal antibody, a purple color formation on the nitrocellulose membrane was detected in the dot-blot test. Biofilm formation abilities of reference V. anguillarum isolates and two field V. anguillarum isolates on ELISA plate were examined. Also, it was detected that only one field isolate has formed pellicle in the liquid-air interface.
Discussion and conclusion
As a result, with this study V. anguillarum was recovered from European sea bass cultured in the fish farms located in the Aegean Sea region and identified by using bacteriological methods. Their serotype was detected as O1 and this result was confirmed with Dot-blot test. Biofilm formation abilities of reference V. anguillarum isolates and two field V. anguillarum isolates on ELISA plate were examined and it was detected that one field isolate has formed pellicle in the liquid-air interface. With this study, biofilm formation ability of V. anguillarum recovered from diseased European sea bass cultured in Turkey was investigated for the first time.