Research Information System
Aquaculture Europe 14, San Sebastian, Spain, 14 - 17 October 2014, pp.193-194
Turkey is one of the leading countries in gilt-head sea bream aquaculture in Mediterranean region. One of the most important obstacles in gilt-head seabream production is the vibriosis disease caused by various Vibrionacea species and resulting in mortalities and important economical losses in aquaculture. Vibrio anguillarum is the most frequently isolated species and it is the major etiological agent of vibriosis. This pathogen was recovered from various cultured marine fish species such as gilt-head seabream, Atlantic salmon, European sea bass and red porgy as well as from rainbow trout cultured in freshwater in Turkey.
Exact identification and characterization of the causative organisms is necessary for the initiation of proper therapy or prophylaxis of vibriosis, as well as for epidemiological investigations. Diagnosis of V. anguillarum is mainly based on the study of the phenotypical traits of the isolated bacteria followed by serological confirmation and determination of the O-serotype of the pathogen. Recently, 23 O-serotypes of this species were determined, however serotype O1, O2 and O3 are mainly associated with fish diseases. Differences of the membrane protein profiles of different serotypes of this species were revealed by using SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) analysis. Also molecular biological studies are targeted for the identification and characterization of this species. A sensitive and specific molecular method (rpoN-PCR) was developed for the detection of this pathogen. Molecular fingerprinting methods such as REP-PCR (repetitive extragenic palindromic-polymerase chain reaction) provide information at or below the subspecies level. The aim of this study is the determination of some phenotypic, serologic and molecular biologic traits and hence the characterization of V. anguillarum isolates recovered from moribund gilt-head sea bream cultured in the Aegean Sea coasts of Turkey.
12 moribund fish samples weighing between 3-80 g, showing the general clinical symptoms of vibriosis were obtained from 2 inland hatcheries and 1 off-shore marine cage facilities located in the Aegean Sea coasts of Turkey. Fish samples were anaesthetized and dissected under aseptic conditions and examined for bacteriology. Bacteriological inoculations from the internal organs of fish samples were streaked onto TSA (2% NaCl) and incubated at 24 °C for 3 days. Creamy colored round colonies with a diameter of 3-4 mm were identified by using routine bacteriological methods and API 20E test strips. Serotype of V. anguillarum isolates were determined by using slide-agglutination test (Sorensen and Larsen, 1986). Membrane proteins were purified as described by Crosa and Hodges (1981) and Santos et al. (1995) and their profiles were determined by SDS-PAGE method and stained with coomassie brilliant blue. For the molecular studies, DNA extraction from fresh bacterial cultures was performed by using InstaGene matrix (BioRad) and later Ready-to-go™ PCR beads (Amersham Pharmacia Biotech) were used for PCR amplification. rpoN-PCR amplification was used for the molecular confirmation of the identification (Forward: rpoN-ang5' Reverse: rpoN-ang3' ) (Gonzalez et al., 2003). REPPCR method was used for the determination of the molecular characterization of the isolates (Forward: REP1D Reverse: REP2D) (Rodriguez et al., 2006). Reference V. anguillarum strains of serotype O1 and O2 from the culture collection of Universidade de Santiago de Compostela (Spain) were used in all steps of this study.
In this study, 5 V. anguillarum isolates were recovered from moribund gilt-head sea bream samples and characterized. They were initially identified depending on their phenotypic and biochemical traits. In this study V. anguillarum isolates formed nearly a homogenous biochemical profile in routine tests but 4 different API 20E profiles were detected. Three isolates made strong agglutination against the antibodies raised against V. anguillarum serotype 01. Other two isolates did not made any agglutination against antibodies raised against the serotype O1, O2 and O3 of this species in any of the three repeats of the test. All field isolates generated similar protein profiles both in whole-cell protein and outer membrane
protein SDS-PAGE analysis. The major outer membrane protein was detected to be 41 kDa in serotype O1 and 38 kDa in serotype O2. The isolates which were negative in slide agglutination test generated similar protein profiles with serotype O1. Hence, the serotype of the isolates which were negative on slide agglutination could be determined by using SDSPAGE. All V. anguillarum isolates used in this study produced a unique, clear 519 bp length band in rpoN-PCR and this result is a molecular confirmation of the bacteriological, serological and immunochemical identification. They showed a slight heterogeneity in REP-PCR analysis and generated 2 different profiles with a difference in one band.
In this study, V. anguillarum infections in cultured gilt-head sea bream were detected and the pathogen was characterized by using biochemical, immunochemical and molecular methods. Slight variations in the biochemical properties of the isolates recovered from different areas of Turkey. Also it was determined that SDS-PAGE can be used as a supplementary method for the identification of the serotype of this pathogen. The success of the molecular methods for the identification and characterization was also determined. In conclusion, the results of this study shows that despite an intense vaccination programme, V. anguillarum is still among the major pathogens of gilthead sea bream cultured in Turkey and detailed studies should be carried out for the characterization of this pathogen for the generation of better protection strategies.