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The abalone and the bacteria: recent progresses in molecular biology

For several years, a bacterium has been decimating wild and farmed populations of European abalone. Molecular biology helped develop a rapid, specific and sensitive technique for detecting and quantifying virulent strains.

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The European abalone is highly esteemed as a delicacy and is fished in wild populations or cultivated in aquaculture farms. But these two types of populations are declining because of serious mortality events caused by parasites or bacteria. Since 1998, recurrent mortality outbreaks occurred at the end of warm season in French farms and field stocks; they were caused by a bacterium, Vibrio harveyi, a common pathogen of many commercially cultured fish and shellfish species worldwide. In the abalone, this infection causes a loss of muscular strength and the appearance of white pustules on the foot, leading to a fatal septicemia and to mortality rates reaching 80% within a few days to three weeks. This pathology affects individuals involved in reproduction or with reduced immune defense capacities, and breaks out only if water temperature exceeds 17°C; this temperature dependence is an important factor in the context of global ocean warming.

However monitoring this pathogen is difficult because it is often present together with other Vibrio species which cannot be reliably identified without gene sequencing techniques, a time consuming approach poorly adapted to perform rapid diagnostic tests in the context of disease surveillance and prevention in aquaculture facilities. The goal of the study was the development and evaluation of a rapid, specific, sensitive and quantitative method for the detection of V. harveyi strains in either pure cultures, in seawater or in the hemolymph (a liquid whose function is analog to that of blood).



The European abalone, Haliotis tuberculata (© CNRS/E. Amice)


The technique called Taqman is a real-time polymerase chain reaction (PCR). The principle of PCR is to replicate in huge proportions very small traces of DNA; it enables to amplify up to detectable concentrations specific genes chosen for their role. Real-time PCR is an extension of this technique where the amplified DNA is detected as the reaction progresses in "real time", thereby allowing the measure of the initial amount of DNA in the sample. It is thus both qualitative (detection of the gene) and quantitative (measure of its abundance).

Two sets of tools specific to this technique were developed. The first one aimed at the detection and quantification of V. harveyi presence through the targeting of a particular region of the housekeeping gene toxR which is specific of this species. The other one allowed the detection and quantification of its virulence through the targeting of gene parA on the suspected virulent pVCR1 plasmid (plasmids are DNA molecules physically separate from chromosomal DNA within a bacterium cell). The amplification of these two DNA sequences was performed simultaneously on 17 bacterial strains.


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Principle of the study (adapted from Renault and Schikorski, 2010)

Results confirm that the gene toxR allows the detection of all V. harveyi strains, including reference strains, whereas the tests are negative with other species. The method also enables to quantify the amount of genes parA present in the sample, with a very good correlation between the number of DNA amplification cycles and the initial bacterial concentration. The size of the DNA fragments was confirmed by the migration of PCR products in agarose gel (electrophoresis).

Visualization of the amplified DNA fragments produced by PCR according to their size (pairs of bases), for genes parA (lane 1) and toxR (lane 2); lane MT shows the scale.

Polymerase chain reaction (PCR) is a very common technique; it is used in most works on a rapid, specific and sensitive molecular detection of species of the genus Vibrio, and in particular V. harveyi. Measuring bacterial concentration in a sample is possible with real-time PCR, but the application of this technique to Vibrio species was so far limited to a few human pathogens such as V. parahaemolyticus (food poisoning) or V. cholerae (cholera). It did not allow the distinction of V. harveyi from phylogenetically related species. This study is the first to adapt it to detect exclusively V. harveyi strains affecting abalone: even with a large number of amplification cycles, no signal was detected for seven closely related species.

The method is also specific for the gene associated to virulence, as the only strains tested positive were those whose experimental injection provokes a high mortality. DNA extraction procedure was satisfactory, allowing a reliable estimation of the amount of infecting DNA in the sample; it has also been shown there are probably no more than 2 to 4 copies of the pCVR1 plasmid in each bacterial cell. The detection limit of V. harveyi with this technique is 3,700 cells/ml of water, which is much higher than for the other Vibrio species (about 200 cells/ml). The sensitivity of the method could be improved by the development of concentration procedures of bacteria before DNA extraction.

If applied to hemolymph (which can be sampled on live animals), this method will be useable in abalone farms to detect V. harveyi and monitor the occurrence and development of mortality episodes, thereby limiting their economic consequences for the producers.


The paper

Schikorski D., Renault T. , Paillard C., Bidault-Toffin A., Tourbiez D., Saulnier D., 2013. Development of TaqMan real-time PCR assays for monitoring Vibrio harveyi infection and a plasmid harbored by virulent strains in European abalone Haliotis tuberculata aquaculture. Aquaculture 392–395 : 106–112.

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The authors

This work was conducted in collaboration by researchers of Lemar (IUEM) and Ifremer (Laboratoire de Génétique et Pathologie, UMR Ecosystèmes Insulaires Océaniens).


The journal

Aquaculture is an international journal published by Elsevier since 1972. Its scope covers all aspects of cultivation or farming of aquatic organisms, either vegetal or animal, in freshwater or marine environments: finfish, crustaceans, mollusks or plants produced for human consumption.



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