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

Abstract

The Gram-negative bacterium Vibrio harveyi is known to be highly pathogenic for the European abalone Haliotis tuberculata, which is a gastronomically important marine gastropod with a high commercial value. Since 1998, some particular bacterial strains are described as implicated in recurrent mortality outbreaks in French farm and field stocks of abalone. Recently, a 9.6kb plasmid named pVCR1, was shown to be harbored by one highly V. harveyi virulent ORM4 strain suggesting its involvement in virulence phenotype. Thus, we have developed in the present study two TaqMan real-time PCR assays allowing to (i) rapidly and specifically detect, by a duplex procedure and in less than 2h, both V. harveyi and the presence of plasmid pVCR1 from unidentified bacterial colony and to (ii) quantify both V. harveyi and the plasmid pVCR1 in the hemolymph of abalone or its surrounding seawater. Quantification curves of V. harveyi or ORM4 strain seeded in hemolymph or artificial sea water samples were equivalent showing excellent qPCR efficacies and detection level as low as 18V. harveyi cell-equivalent genomic DNA in a PCR reaction well. This qPCR allowed us to monitor V. harveyi ORM4 strain in experimentally infected H. tuberculata. These diagnosis assays could provide powerful and useful tools to better understand the epidemiology of vibriosis caused by V. harveyi in different cultured marine species including H. tuberculata.